Curcumin analogues as zinc chelators and their uses

ABSTRACT

This invention provides a compound having the structure 
                         
wherein α, β, X, Y, and R 1 -R 11  are defined herein. This invention also provides a pharmaceutical composition comprising the above compounds, a method of inhibiting the activity and/or levels of a matrix metalloproteinase (MMP), a method of inhibiting the production of a cytokine in a population of cells, a method of inhibiting the production of a growth factor in a population of cells, and a method of inhibiting NFκ-B activation in a population of cells.

This application is a §371 National Stage of PCT InternationalApplication No. PCT/US2010/034971, filed May 14, 2010, claiming priorityof Provisional Application No. 61/216,392, filed May 15, 2009, thecontents of which are hereby incorporated by reference into thisapplication.

This application claims the benefit of U.S. Provisional Application No.61/216,392, filed May 15, 2009, the content of which is herebyincorporated by reference in its entirety.

Throughout this application, certain publications are referenced inparentheses. Full citations for these publications may be foundimmediately preceding the claims. The disclosures of these publicationsin their entireties are hereby incorporated by reference into thisapplication in order to describe more fully the state of the art towhich this invention relates.

BACKGROUND OF THE INVENTION

For decades, soon after the first of the matrix metalloproteinases(MMPs), known as collagenase-1 or MMP-1, was discovered in the early1960s (1), academics and industry have been trying to develop safe andeffective pharmaceuticals to inhibit these calcium- and zinc-dependentneutral proteinases which are expressed and activated in excessivelevels during a variety of diseases (see (2) and (3) for reviews). Therationale for this drug development strategy lies in the fact thatcollagen and the other connective tissue constituents: (a) arecollaboratively degraded by these MMPs (now numbering more than 25genetically distinct types, “22 found in the human genome” (2)); and (b)are found virtually everywhere in the body (e.g., skin, bone, tendonsand ligaments, cornea of the eye, cartilage of the joints), and theexcessive destruction of these constituents is a key event in thepathogenesis of numerous diseases ranging from inflammatory conditions(e.g., rheumatoid arthritis, atherosclerosis, periodontitis), tometabolic bone diseases (e.g., postmenopausal osteoporosis,diabetes-induced osteopenia), to cancer invasion, metastasis andangiogenesis (2, 4).

Matrix metalloproteinases (MMPs) are a collective of over thirtyzinc-containing endopeptidases that include the gelatinases,stromelysins, and collagenases, released as inactive zymogens andbecoming active only when the propeptide is cleaved (5). The gelatinasesinclude MMP-2 and MMP-9, and the stromelysins include MMP-3, -7, 10, and-11. The collagenases include MMP-1, -8, and -13 (5). The MMPs, whenconstitutively expressed or induced by pro-inflammatory agents, such ascytokines, hormones, bacterial products, endotoxins, among others, candegrade all components of the extracellular matrix (5). Underphysiological conditions, MMPs are regulated by endogenous inhibitors,particularly the tissue inhibitors of metalloproteinases (TIMPs) (5,6a).

Aberrant MMP activity and expression has been implicated in a number ofpathological conditions, including rheumatoid arthritis (RA),osteoarthritis (OA), metastases, periodontal disease, angiogenesis,emphysema, multiple sclerosis (5), and cardiovascular disease, such asatherosclerosis, myocardial infarction, arterial restenosis afterangioplasty and aneurysm development (6a). Recent research has alsoimplicated MMPs in asthma attacks, chronic obstructive pulmonarydisease, and premature skin aging (6a) and inflammatory skin disease(6b, 6c). Their involvement in the epidermal growth factor-receptoractivation pathway leading to cardiac hypertrophy has also been reported(6a). It is believed that an imbalance between the active enzymes andtheir natural inhibitors leads to the accelerated destruction ofconnective tissue and the potential for using specific enzyme inhibitorsas therapeutic agents to redress this balance has led to intensiveresearch focused on the design, synthesis and molecular deciphering oflow-molecular-mass inhibitors of this family of proteins (7).

At least 56 MMP inhibitors have been pursued as clinical candidatessince the late 1970's, and as of 2006, only 1 inhibitor, asub-antimicrobial (low-dose) doxycycline formulation (Periostat® forperiodontal disease), has been approved (8). Early clinical studies withother known MMP inhibitors, in particular a series of hydroxamic acids,have revealed a severe adverse side-effect frequently referred to as themusculoskeletal syndrome (MSS), which is a tendonitis-like fibromyalgia(8). In subsequent MMP inhibitor clinical trials, efforts to avoid MSScoupled with an inability to assess the therapeutic index (ie., theratio between the dose required for efficacy vs. toxicology), may haveresulted in dose selection beneath the minimal effective dose, hamperingMMP inhibitor development (8).

Therefore, there is a need for the development of new MMP inhibitors.

Curcumin (diferuloylmethane, FIG. 1), the major component incurcuma/turmeric, is an antioxidant polyphenol from the plant Curcumalonga and is commonly used as a spice component. Curcumin has been usedto treat inflammation and exerts antiproliferative and proapoptoticeffects against various tumors in vitro and in vivo, and it has beenfound to suppress carcinogenesis of the breast and other organs (9, 10).Bachmeier and coworkers have reported downregulation of the inflammatorycytokines CXCL1 and CXCL2 in breast cancer cells via NFκB (9). Oralcurcumin efficacy in vivo has been shown in models for many conditionswith oxidative damage and inflammation, including many types of cancer,diabetes, atherosclerosis, arthritis, stroke, peripheral neuropathy,inflammatory bowel disease, and brain trauma (11). Curcumin, along withits tetrahydro derivative, tetrahydrocurcumin (THC, FIG. 1.), has beenshown to inhibit IL-1β in an acute brain inflammation model whilecurcumin was more effective than THC in attenuating plaque pathogenesisin studies of curcumin efficacy in models of neuroinflammation, which isimplicated in the pathogenesis of many neurodegenerative disorders,including Alzheimer's disease (AD) (11).

Curcumin has also been shown to inhibit MMPs. Curcumin (at 15 μMconcentration) has been observed to exert a significant inhibitoryeffect on MMP-2 activity which was not reversible even after cells weregrown for 28 days without curcumin (12). It is known that highlymetastatic cells become less aggressive when MMP-2 expression oractivity is reduced and previous studies have also shown that curcuminreduces MMP-2 expression in breast carcinoma cell lines (12). Thisreduction of MMP-2 activity could be an important reason foranti-metastatic property of curcumin (12). In addition, curcumin hasalso been shown to inhibit MMP-9 expression in human astroglioma cells(13). Analogues and derivatives of curcumin have previously beendescribed for use against various cancers (14, 15, 16, 17) as well aspancreatitis (18).

While curcumin has been shown to have multiple beneficial effects, poororal absorption of curcumin in both humans and animals has raisedseveral concerns that this may limit its clinical impact (11).

Herein, novel chemically-modified curcumins as inhibitors of matrixmetalloproteinases and pro-inflammatory cytokine production aredisclosed.

SUMMARY OF THE INVENTION

This invention provides a compound having the structure

wherein

bond α and β are each, independently, present or absent;

X is CR₅ or N; Y is CR₁₀ or N;

R₁ is H, CF₃, halogen, —NO₂, —OCF₃, —OR₁₂, —NHCOR₁₂, —SR₁₂, —SO₂R₁₃,—COR₁₄, —CSR₁₄, —CNR₁₄, —C(═NR₁₂)R₁₄, —C(═NH)R₁₄, —SOR₁₂, —POR₁₂,—P(═O)(OR₁₂)(OR₁₃), or —P(OR₁₂)(OR₁₃),

-   -   wherein R₁₂ and R₁₃ are each, independently, H, C₁₋₁₀ alkyl,        C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;    -   R₁₄ is C₂₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroaryl,        heterocyclyl, methoxy, —OR₁₅, —NR₁₆R₁₇, or

-   -    wherein R₁₅ is H, C₃₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl;        -   R₁₆ and R₁₇ are each, independently, H, alkyl, C₂₋₁₀            alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;        -   R₁₈, R₁₉, R₂₁, and R₂₂ are each independently H, halogen,            —NO₂, —CN, —NR₂₃R₂₄, —SR₂₃, —SO₂R₂₃, —CO₂R₂₃, —OR₂₅, CF₃,            —SOR₂₃, —POR₂₃, —C(═S)R₂₃, —C(═NH)R₂₃, C(═NR₂₄)R₂₃,            —C(═N)R₂₃, —P(═O)(OR₂₃)(OR₂₄), —P(OR₂₃)(OR₂₄), —C(═S)R₂₃,            C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl,            or heterocyclyl;            -   wherein R₂₃, R₂₀, and R₂₅ are each, independently, H,                C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,                heteroaryl, or heterocyclyl;        -   R₂₀ is halogen, —NO₂, —CN, —NR₂₆R₂₇, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀            alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;            -   wherein R₂₆ and R₂₇ are each, independently, H, C₁₋₁₀                alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl,                or heterocyclyl;

R₂, R₃, R₃, R₅, R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are each independently, H,halogen, —NO₂, —CN, —NR₂₈R₂₉, —NHR₂₈R₂₉ ⁺, —SR₂₉, —SO₂R₂₈, —OR₂₈,—CO₂R₂₈, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,heteroaryl, or heterocyclyl;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, or —C(═O)-heterocyclyl; and

wherein when R₁ is H, R₃, R₄, R₅, R₈, R₉, or R₁₀, is halogen, —NO₂, —CN,—NR₂₈R₂₉, —NHR₂₈R₂₉ ⁺, —SR₂₈, —SO₂R₂₈, —CO₂R₂₈, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, or —C(═O)-heterocyclyl; and

wherein each occurrence of alkyl, alkenyl, or alkynyl is branched orunbranched, unsubstituted or substituted;

or a salt thereof.

This invention provides a pharmaceutical composition comprising apharmaceutically acceptable carrier and of the above compounds.

This invention also provides a method of inhibiting the activity and/orlevels of a matrix metalloproteinase (MMP) comprising contacting thematrix metalloproteinase or a cell producing an MMP or MMPs with any oneof the above compounds so as to inhibit the activity of a matrixmetalloproteinase.

This invention further provides a method of inhibiting the production ofa cytokine in a population of mammalian cells comprising contacting thepopulation of cells with any one of the above compounds so as to inhibitproduction of a cytokine.

This invention yet further provides a method of inhibiting theproduction of a growth factor in a population of mammalian cellscomprising contacting the population of cells with the any one of theabove compounds so as to inhibit production of a growth factor.

This invention provides a method of inhibiting NFκ-B activation in apopulation of cells comprising contacting the population of cells withany one of the above compounds so as to inhibit NFκ-B activation.

This invention provides a method of increasing water solubility, metalbinding ability, MMP inhibition activity, cytokine inhibition activity,growth factor inhibition activity, or NFκB activation inhibitionactivity of curcumin comprising synthesizing a compound having thestructure

wherein

bond α and β are each, independently, present or absent;

X is CR₅ or N; Y is CR₁₀ or N;

R₁ is H or an electron-withdrawing group;

R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are each independently, H,halogen, —NO₂, —CN, —NR₂₈R₂₉, —NHR₂₉R₂₉ ⁺, —SR₂₈, —SO₂R₂₈, —OR₂₈,—CO₂R₂₈, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,heteroaryl, or heterocyclyl;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, or —C(═O)-heterocyclyl; and

wherein when R₁ is H, R₃, R₄, R₅, R₈, R₉, or R₁₀, is halogen, —NO₂, —CN,—NR₂₈R₂₉, —NHR₂₈R₂₉ ⁺, —SR₂₈, —SO₂R₂₈, —CO₂R₂₈, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, or —C(═O)-heterocyclyl; and

wherein each occurrence of alkyl, alkenyl, or alkynyl is branched orunbranched, unsubstituted or substituted;

or a salt thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Chemical structures of curcumin, tetrahydrocurcumin (THC), and1,10-O-phenanthroline.

FIG. 2. Cytokine levels in conditioned media (CM) of monocytes: Effectof compound 1.

FIG. 3. Development of type 1 diabetes rat model. Body weight changes:control vs. diabetes at day 21. 16% differential observed at 3 weeks.

FIG. 4. Blood glucose levels at day 21: control vs. diabetes.

FIG. 5. A comparison of the MMP-8 inhibitory potency (IC₅₀) of severalcurcumin compounds & 1,10-phenanthroline.

FIG. 6. A comparison of the MMP-9 Inhibitory Potency (IC₅₀) of severalchemically-modified curcumins and 1,10-phenanthroline

FIG. 7. Effect of compound 1 on blood glucose and MMPs. Diabetesincreases MMP-2 & MMP-9 in rat plasma. * “Low-dose” reflects low serumconcentration (˜0.1 μg/ml) and short duration (1 week treatment);NDC=non-diabetic Control (n=4 rats/group); UD=Untreated Diabetic (n=6rats/group); D+compound 1=Diabetic treated with compound 1 (n=6rats/group).

FIG. 8. MMP-8 levels, assessed by Western blots, in partially purifiedrat skin extract.

FIG. 9. The Effect of Diabetes and Orally-Administered Compound 1 on (A)Blood Glucose and (B) Hemoglobin Alc levels.

FIG. 10. The Effect of Diabetes and Oral Administration of Compound 1 onGelatinase Levels in Gingiva, Assessed by Gelatin Zymography. Each valuerepresents a pool of gingival tissue from 3 rats/experimental group.

FIG. 11. The Effect of Diabetes and Oral Administration of Compound 1 onIL-1β levels by ELISA.

FIG. 12. Diabetes Increases the Levels of MMP-2 (pro- and active-forms)in the skin of rats, when compared to MMP-2 levels in the skin ofnon-diabetic control rats, assessed by gelatin zymography.

FIG. 13. The effect of oral administration of compound 1 on alveolar(periodontal) bone loss in hyperglycemic type I diabetic rats. Eachvalue represents the mean bone loss score±the standard Error of the Mean(S.E.M.)

FIG. 14. Effect of MMP inhibitors on H₂O₂ (100 μM, 6 h) induced LDHrelease in neonatal rat ventricular myocytes (n=4).

FIG. 15. Effect of MMP inhibitors on H₂O₂ (100 μM, 24 h) induced LDHrelease in neonatal rat ventricular myocytes (n=4).

FIG. 16. Effect of MMP inhibitors on doxorubicin (0.5 μM, 6 h) inducedcaspase-3 activity in neonatal rat ventricular myocytes (n=4).

FIG. 17. Effect of MMP inhibitors on doxorubicin (0.5 μM, 24 h) inducedcaspase-3 activity in neonatal rat ventricular myocytes (n=4).

FIG. 18. Analysis of ³⁵SO₄ ⁻ release following treatment with curcuminderivatives (24 h).

FIG. 19. Analysis of ³⁵SO₄ ⁻ release following treatment with curcuminderivatives (48 h).

FIG. 20. Analysis of ³⁵SO₄ ⁻ release following treatment with curcuminderivatives (72 h).

FIG. 21. Total ³⁵SO₄ ⁻ release following treatment with curcuminderivatives (24 hr pretreatment+24 h treatment).

FIG. 22. Mean ³⁵SO₄ ⁻ release following treatment with curcuminderivatives (24 hr pretreatment+24 h treatment).

FIG. 23-25. Gelatin zymography (in the presence of 2 or 10 mMCa⁺⁺) andWestern blot (both assays used purified MMP-9, 92 kDa gelatinase, as astandard). The 10 μM and 20 μM concentrations of compound 1 inhibitedthe production and/or activity of MMP-9 generated by the human prostatecancer cells.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a compound having the structure

wherein

bond α and β are each, independently, present or absent;

X is CR₅ or N; Y is CR₁₀ or N;

R₁ is H, CF₃, halogen, —NO₂, —OCF₃, —OR₁₂, —NHCOR₁₂, —SR₁₂, —SO₂R₁₃,—CSR₁₄, —CNR₁₄, —C(═NR₁₂)R₁₄, —C(═NH)R₁₄, —SOR₁₂, —POR₁₂,—P(═O)(OR₁₂)(OR₁₃), or —P(OR₁₂)(OR₁₃),

-   -   wherein R₁₂ and R₁₃ are each, independently, H, C₁₋₁₀ alkyl,        C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;    -   R₁₄ is C₂₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroaryl,        heterocyclyl, methoxy, —OR₁₅, —NR₁₆R₁₇, or

-   -    wherein R₁₅ is H, C₃₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl;        -   R₁₆ and R₁₇ are each, independently, H, C₁₋₁₀ alkyl, C₂₋₁₀            alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;        -   R₁₈, R₁₉, R₂₁, and R₂₂ are each independently H, halogen,            —NO₂, —CN, —NR₂₃R₂₄, —SR₂₃, —SO₂R₂₃, —CO₂R₂₃, —OR₂₅, CF₃,            —SOR₂₃, —POR₂₃, —C(═S)R₂₃, —C(═NH)R₂₃, —C(═NR₂₄)R₂₃,            —P(═O)(OR₂₃)(OR₂₄), —P(OR₂₃)(OR₂₄), —C(═S)R₂₃, C₁₋₁₀ alkyl,            C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or            heterocyclyl;            -   wherein R₂₃, R₂₄, and R₂₅ are each, independently, H,                C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,                heteroaryl, or heterocyclyl;        -   R₂₀ is halogen, —NO₂, —CN, —NR₂₆R₂₇, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀            alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;            -   wherein R₂₆ and R₂₇ are each, independently, H, C₁₋₁₀                alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl,                or heterocyclyl;

R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are each independently, H,halogen, —NO₂, —CN, —NR₂₈R₂₉, —NHR₂₈R₂₉ ⁺, —SR₂₈, —SO₂R₂₈, —OR₂₈,—CO₂R₂₈, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,heteroaryl, or heterocyclyl;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, or —C(═O)-heterocyclyl; and

wherein when R₁ is H, R₃, R₄, R₅, R₈, R₉, or R₁₀, is halogen, —NO₂, —CN,—NR₂₈R₂₉, —NHR₂₈R₂₉ ⁺, —SR₂₈, —SO₂R₂₈, —CO₂R₂₈, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, or —C(═O)-heterocyclyl; and

wherein each occurrence of alkyl, alkenyl, or alkynyl is branched orunbranched, unsubstituted or substituted;

or a salt thereof.

In an embodiment, when R₁ is H, R₃, R₄, R₅, R₈, R₉, or R₁₀, is —NO₂,—NR₂₈R₂₉, —NHR₂₈R₂₉ ⁺, —SR₂₈, —SR₂₈, —SO₂R₂₈, —CO₂R₂₈, CF₃, C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl; or a salt thereof.

In another embodiment, when R₁ is H, R₄ or R₉ is —NO₂, —NR₂₈R₂₉,—NHR₂₈R₂₉ ⁺; or a salt thereof.

In yet another embodiment, when R₁ is H, R₄ or R₉ is —NR₂₈R₂₉ or—NHR₂₈R₂₉ ⁺; or a salt thereof.

In an embodiment,

R₁ is H or —COR₁₄,

-   -   wherein R₁₄ is methoxy or —NH-phenyl;

R₂, R₅, R₆, R₇, R₁₀ and R₁₄ are each H;

R₃, R₄, R₈, and R₉ are each, independently H, —OH, —OCH₃, —N(CH₃)₂ or—NH(CH₃)₂;

or a salt thereof.

In an embodiment, the compound has the structure

or a salt thereof.

In an embodiment, the compound has the structure

where

bond α and β are each, independently, present or absent;

R₁ is CF₃, halogen, —NO₂, —OCF₃, —OR₁₂, —NHCOR₁₂, —SR₁₂, —SO₂R₁₃,—COR₁₄, —CSR₁₄, or —CNR₁₄,

-   -   wherein R₁₂ and R₁₃ are each, independently, H, C₁₋₁₀ alkyl,        C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;    -   R₁₄ is C₂₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroaryl,        heterocyclyl, methoxy, —OR₁₅, —NR₁₆R₁₇, or

-   -    wherein R₁₅ is H, C₃₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl;        -   R₁₆ and R₁₇ are each, independently, H, C₁₋₁₀ alkyl, C₂₋₁₀            alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;        -   R₁₈, R₁₉, R₂₁, and R₂₂ are each independently H, halogen,            —NO₂, —CN, —NR₂₃R₂₄, —SR₂₃, —SO₂R₂₃, —CO₂R₂₃, —OR₂₅, CF₃,            C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl,            or heterocyclyl;            -   wherein R₂₃, R₂₄, and R₂₅ are each, independently, H,                C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,                heteroaryl, or heterocyclyl;        -   R₂₀ is halogen, —NO₂, —CN, —NR₂₆R₂₇, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀            alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;            -   wherein R₂₆ and R₂₇ are each, independently, H, C₁₋₁₀                alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl,                or heterocyclyl;

R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are each independently, H,halogen, —NO₂, —CN, —NR₂₈R₂₉, —SR₂₈, —SO₂R₂₈, —OR₂₈, —CO₂R₂₈, CF₃, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, or C₂₋₁₀ alkynyl; and

wherein each occurrence of alkyl, alkenyl, or alkynyl is branched orunbranched, unsubstituted or substituted; and

wherein at least one of R₂, R₃, R₄, R₅, and R₆ and at least one of R₇,R₈, R₉, R₁₀, and R₁₁, are each, independently, —OR₂₈;

or a salt thereof.

In an embodiment, the compound has the structure

wherein R₁ is CF₃, halogen, —NO₂, —OCF₃, —OR₁₂, —NHCOR₁₂, —SR₁₂,—SO₂R₁₃, —COR₁₄, —CSR₁₄, or —CNR₁₄,

-   -   wherein R₁₂ and R₁₃ are each, independently, H, C₁₋₁₀ alkyl,        C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;    -   R₁₄ is C₂₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroaryl,        heterocyclyl, methoxy, —OR₁₅, —NR₁₆R₁₇, or

-   -    wherein R₁₅ is H, C₃₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl;        -   R₁₆ and R₁₇ are each, independently, H, C₁₋₁₀ alkyl, C₂₋₁₀            alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;        -   R₁₈, R₁₉, R₂₁, and R₂₂ are each independently H, halogen,            —NO₂, —CN, —NR₂₃R₂₄, —SR₂₃, —SO₂R₂₃, —CO₂R₂₃, —OR₂₅, CF₃,            C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl,            or heterocyclyl;            -   wherein R₂₃, R₂₄, and R₂₅ are each, independently, H,                C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,                heteroaryl, or heterocyclyl;        -   R₂₀ is halogen, —NO₂, —CN, —NR₂₆R₂₇, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀            alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;            -   wherein R₂₆ and R₂₇ are each, independently, H, C₁₋₁₀                alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl,                or heterocyclyl;

R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are each independently, H,halogen, —NO₂, —CN, —NR₂₈R₂₉, —SR₂₈, —SO₂R₂₈, —OR₂₈, —CO₂R₂₉, CF₃, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, or C₂₋₁₀ alkynyl; and

wherein each occurrence of alkyl, alkenyl, or alkynyl is branched orunbranched, unsubstituted or substituted; and

wherein at least one of R₂, R₃, R₃, R₅, and R₆ and at least one of R₇,R₈, R₉, R₁₀, and R₁₁, are each, independently, —OR₂₈;

or a salt thereof.

In another embodiment, the compound has the structure

wherein R₁₄ is C₂₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroaryl,heterocyclyl, methoxy, —OR₁₅, —NR₁₅R₁₇, or

-   -   wherein R₁₅ is H, C₃₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl;    -   R₁₆ and R₁₇ are each, independently, H, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;    -   R₁₈, R₁₉, R₂₁, and R₂₂ are each independently H, halogen, —NO₂,        —CN, —NR₂₃R₂₄, —SR₂₃, —SO₂R₂₃, —CO₂R₂₃, —OR₂₅, CF₃, C₁₋₁₀ alkyl,        C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;        -   wherein R₂₃, R₂₄, and R₂₅ are each, independently, H, C₁₋₁₀            alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or            heterocyclyl;    -   R₂₀ is halogen, —NO₂, —CN, —NR₂₆R₂₇, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;        -   wherein R₂₆ and R₂₇ are each, independently, H, C₁₋₁₀ alkyl,            C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or            heterocyclyl;

R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are each independently, H,halogen, —NO₂, —CN, —NR₂₈R₂₉, —SR₂₈, —SO₂R₂₈, —OR₂₈, —CO₂R₂₈, CF₃, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, or C₂₋₁₀ alkynyl; and

wherein each occurrence of alkyl, alkenyl, or alkynyl is branched orunbranched, unsubstituted or substituted; and

wherein at least one of R₂, R₃, R₄, R₅, and R₆ and at least one of R₇,R₈, R₉, R₁₀, and R₁₁, are each, independently, —OR₂₈;

or a salt thereof.

In yet another embodiment, the compound has the structure

wherein R₁₄ is C₂₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroaryl,heterocyclyl, methoxy, —OR₁₅, —NR₁₆R₁₇, or

-   -   wherein R₁₅ is H, C₃₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl;    -   R₁₆ and R₁₇ are each, independently, H, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;    -   R₁₈, R₁₉, R₂₁, and R₂₂ are each independently H, halogen, —NO₂,        —CN, —NR₂₃R₂₄, —SR₂₃, —SO₂R₂₃, —CO₂R₂₃, —OR₂₅, CF₃, C₁₋₁₀ alkyl,        C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;        -   wherein R₂₃, R₂₄, and R₂₅ are each, independently, H, C₁₋₁₀            alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or            heterocyclyl;    -   R₂₀ is halogen, —NO₂, —CN, —NR₂₆R₂₇, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;        -   wherein R₂₆ and R₂₇ are each, independently, H, C₁₋₁₀ alkyl,            C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or            heterocyclyl;

R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are each independently, H,halogen, —NO₂, —CN, —NR₂₈R₂₉, —SR₂₈, —SO₂R₂₈, —OR₂₈, —CO₂R₂₈, CF₃;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₅ alkyl, C₂₋₅ alkenyl,        or C₂₋₅ alkynyl; and

wherein each occurrence of alkyl, alkenyl, or alkynyl is branched orunbranched, unsubstituted or substituted; and

wherein at least one of R₂, R₃, R₄, R₅, and R₆ and at least one of R₇,R₈, R₉, R₁₀, and R₁₁, are each, independently, —OR₂₈;

or a salt thereof.

In an embodiment,

-   -   R₁₄ is methoxy, —OR₁₅ or —NR₁₆R₁₇,    -   wherein R₁₅ is H, C₃₋₁₀ alkyl, C₂₋₁₀ alkenyl, or C₂₋₁₀ alkynyl;    -   R₁₆ and R₁₇ are each, independently, H, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;    -   or a salt thereof.

In another embodiment of the compound, R_(H) is methoxy or —NR₁₆R₁₇,

-   -   wherein R₁₆ and R₁₇ are each, independently, H, C₁₋₁₀ alkyl,        C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;    -   or a salt thereof.

In an embodiment, R₁₄ is —OR₁₅, wherein R₁₅ is H, C₃₋₁₀ alkyl, C₂₋₁₀alkenyl, or C₂₋₁₀ alkynyl; or a salt thereof.

In an embodiment,

R₁₄ is —NR₁₆R₁₇,

-   -   wherein R₁₆ and R₁₇ are each, independently, H or aryl;

R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are each independently, H,—NR₂₈R₂₉, or —OR₂₈,

-   -   wherein R₂₈ and R₂₉ are each, H or C₁₋₁₀ alkyl; or a salt        thereof.

In an embodiment,

R₁₄ is —NH-phenyl;

R₂, R₅, R₆, R₇, R₁₀, and R₁₁ are each H;

R₃, R₄, R₈, and R₉ are each, independently, H, —OH, or —OCH₃; or a saltthereof.

In another embodiment, the compound has the structure

or a salt thereof.

In an embodiment, the compound has the structure

wherein R₃, R₄, R₈, and R₉ are H, —OCH₃, or —OH; R₁₄ is methoxy or—N(CH₃)₂; or a salt thereof.

In another embodiment, the compound has the structure

or a salt thereof.

In an embodiment,

R₁₄ is methoxy;

R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are each, independently, H,—OH, —OCH₃, —NO₂, or —CO₂CH₃; or a salt thereof.

In another embodiment, the compound has the structure

or a salt thereof.

In another embodiment, X is N; or a salt thereof.

In an embodiment, α and β are both present; or a salt thereof.

In another embodiment, the compound has the structure:

or a salt thereof.

This invention provides a pharmaceutical composition comprising apharmaceutically acceptable carrier and of the above compounds.

This invention also provides a method of inhibiting the activity and/orlevels of a matrix metalloproteinase (MMP) comprising contacting thematrix metalloproteinase or a cell producing an MMP or MMPs with any oneof the above compounds so as to inhibit the activity of a matrixmetalloproteinase.

In an embodiment, the matrix metalloproteinase is MMP-1, MMP-2, MMP-3,MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, or MMP-14.

This invention further provides a method of inhibiting the production ofa cytokine in a population of mammalian cells comprising contacting thepopulation of cells with any one of the above compounds so as to inhibitproduction of a cytokine.

In an embodiment, the population of cells is a population of humancells.

In another embodiment, the cytokine is THE-α, IL-1β, MCP-1, IL-8, orIL-6.

In yet another embodiment, the production of a cytokine is induced by anendotoxin, lipopolysaccharide (LPS), a hormone, a cholesterol complex,or an inflammatory mediator, including but not limited to nitric oxide,and reactive oxygen species.

This invention yet further provides a method of inhibiting theproduction of a growth factor in a population of mammalian cellscomprising contacting the population of cells with the any one of theabove compounds so as to inhibit production of a growth factor.

In an embodiment, the growth factor is VEGF, PDGF, TGF-β, or MIP1α.

This invention provides a method of inhibiting NFκ-B activation in apopulation of cells comprising contacting the population of cells withthe any one of the above compounds so as to inhibit NFκ-B activation.

In an embodiment, the population of cells is a population of humancells.

This invention provides a method of increasing water solubility, metalbinding ability, MMP inhibition activity, cytokine inhibition activity,growth factor inhibition activity, or NFκB activation inhibitionactivity of curcumin comprising synthesizing a compound having thestructure

wherein

bond α and β are each, independently, present or absent;

X is CR₅ or N; Y is CR₁₀ or N;

R₁ is H or an electron-withdrawing group;

R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are each independently, H,halogen, —NO₂, —CN, —NR₂₈R₂₉, —NHR₂₈R₂₉ ⁺, —SR₂₈, —SO₂R₂₈, —OR₂₈,—CO₂R₂₈, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,heteroaryl, or heterocyclyl;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, or —C(═O)-heterocyclyl; and

wherein when R₁ is H, R₃, R₄, R₅, R₈, R₉, or R₁₀, is halogen, —NO₂, —CN,—NR₂₈R₂₉, —NHR₂₈R₂₉ ⁺, —SR₂₈, —SO₂R₂₈, CO₂R₂₈, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, or —C(═O)-heterocyclyl; and

wherein each occurrence of alkyl, alkenyl, or alkynyl is branched orunbranched, unsubstituted or substituted;

or a salt thereof.

In an embodiment, the compound synthesized has the structure

wherein

bond α and β are each, independently, present or absent;

R₁ is an electron-withdrawing group;

R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are each independently, H,halogen, —NO₂, —CN, —NR₂₈R₂₉, —SR₂₈, —SO₂R₂₈, —OR₂₈, —CO₂R₂₈, CF₃, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, or C₂₋₁₀ alkynyl; and

wherein each occurrence of alkyl, alkenyl, or alkynyl is branched orunbranched, unsubstituted or substituted; and

wherein at least one of R₂, R₃, R₄, R₅, and R₆ and at least one of R₇,R₈, R₉, R₁₀, and R₁₁, are each, independently, —OR₂₈;

or a salt thereof.

In an embodiment, the compound synthesized has the structure

wherein

R₁ is an electron-withdrawing group;

R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are each independently, H,halogen, —NO₂, —CN, —NR₂₈R₂₉, —SR₂₈, —SO₂R₂₈, —OR₂₈, —CO₂R₂₈, CF₃, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;

-   -   wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀        alkenyl, or C₂₋₁₀ alkynyl; and

wherein each occurrence of alkyl, alkenyl, or alkynyl is branched orunbranched, unsubstituted or substituted; and

wherein at least one of R₂, R₃, R₄, R₅, and R₆ and at least one of R₇,R₈, R₉, R₁₀, and R₁₁, are each, independently, —OR₂₈;

or a salt thereof.

In another embodiment, the compound synthesized has the structure

wherein R₁ is an electron-withdrawing group.

It is understood that the structures described in the embodiments of themethods hereinabove can be the same as the structures of the compoundsdescribed hereinabove.

As used herein, the term “activity” refers to the activation,production, expression, synthesis, intercellular effect, and/orpathological or aberrant effect of the referenced molecule, eitherinside and/or outside of a cell. Such molecules include, but are notlimited to, cytokines, enzymes, growth factors, pro-growth factors,active growth factors, and pro-enzymes. Molecules such as cytokines,enzymes, growth factors, pro-growth factors, active growth factors, andpro-enzymes may be produced, expressed, or synthesized within a cellwhere they may exert an effect. Such molecules may also be transportedoutside of the cell to the extracellular matrix where they may induce aneffect on the extracellular matrix or on a neighboring cell. It isunderstood that activation of inactive cytokines, enzymes andpro-enzymes may occur inside and/or outside of a cell and that bothinactive and active forms may be present at any point inside and/oroutside of a cell. It is also understood that cells may possess basallevels of such molecules for normal function and that abnormally high orlow levels of such active molecules may lead to pathological or aberranteffects that may be corrected by pharmacological intervention.

The term “cytokine” as used herein refers to a cellular signalingmolecule, which includes, but is not limited to, a lymphokine, amonokine, a chemokine, an interferon, an interleukin, or a hormone.Examples of a monokine include, but are not limited to, TNF-α and TNF-β.Examples of a chemokine include, but are not limited to, MCP-1, MCP-2,and MCP-3. Examples of an interferon include, but are not limited to,IFN-α, IFN-β, and IFN-γ. Examples of an interleukin include, but are notlimited to, IL-1α, IL1-β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-10, and IL-12.

The term “growth factor” as used herein refers to a protein that bindsto receptors on the cell surface, with the primary function ofregulating cellular proliferation and/or differentiation. Examples of agrowth factor include, but are not limited to, G-CSF, GM-CSF, MIP1α,MIP1β, TGF-α, TGF-β, VEGF, and PDGF.

The compounds of the present invention include all hydrates, solvates,and complexes of the compounds used by this invention. If a chiralcenter or another form of an isomeric center is present in a compound ofthe present invention, all forms of such isomer or isomers, includingenantiomers and diastereomers, are intended to be covered herein.Compounds containing a chiral center may be used as a racemic mixture,an enantiomerically enriched mixture, or the racemic mixture may beseparated using well-known techniques and an individual enantiomer maybe used alone. The compounds described in the present invention are inracemic form or as individual enantiomers. The enantiomers can beseparated using known techniques, such as those described in Pure andApplied Chemistry 69, 1469-1474, (1997) IUPAC. In cases in whichcompounds have unsaturated carbon-carbon double bonds, both the cis (Z)and trans (E) isomers are within the scope of this invention.

The compounds of the subject invention may have spontaneous tautomericforms. In cases wherein compounds may exist in tautomeric forms, such asketo-enol tautomers, each tautomeric form is contemplated as beingincluded within this invention whether existing in equilibrium orpredominantly in one form.

In the compound structures depicted herein, hydrogen atoms are not shownfor carbon atoms having less than four bonds to non-hydrogen atoms.However, it is understood that enough hydrogen atoms exist on saidcarbon atoms to satisfy the octet rule.

As used herein, “alkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms and may be unsubstituted or substituted. Thus, C₁-C_(n) asin “C₁-C_(n) alkyl” is defined to include groups having 1, 2, . . . ,n−1 or n carbons in a linear or branched arrangement. For example,C₁-C₆, as in “C₁-C₆ alkyl” is defined to include groups having 1, 2, 3,4, 5, or 6 carbons in a linear or branched arrangement, and specificallyincludes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, pentyl,hexyl, and octyl.

As used herein, “alkenyl” refers to a non-aromatic hydrocarbon radical,straight or branched, containing at least 1 carbon to carbon doublebond, and up to the maximum possible number of non-aromaticcarbon-carbon double bonds may be present, and may be unsubstituted orsubstituted. For example, “C₂-C₆ alkenyl” means an alkenyl radicalhaving 2, 3, 4, 5, or 6 carbon atoms, and up to 1, 2, 3, 4, or 5carbon-carbon double bonds respectively. Alkenyl groups include ethenyl,propenyl, butenyl and cyclohexenyl.

The term “alkynyl” refers to a hydrocarbon radical straight or branched,containing at least 1 carbon to carbon triple bond, and up to themaximum possible number of non-aromatic carbon-carbon triple bonds maybe present, and may be unsubstituted or substituted. Thus, “C₂-C₆alkynyl” means an alkynyl radical having 2 or 3 carbon atoms and 1carbon-carbon triple bond, or having 4 or 5 carbon atoms and up to 2carbon-carbon triple bonds, or having 6 carbon atoms and up to 3carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl andbutynyl.

“Alkylene”, “alkenylene” and “alkynylene” shall mean, respectively, adivalent alkane, alkene and alkyne radical, respectively. It isunderstood that an alkylene, alkenylene, and alkynylene may be straightor branched. An alkylene, alkenylene, and alkynylene may beunsubstituted or substituted.

As used herein, “aryl” is intended to mean any stable monocyclic,bicyclic or polycyclic carbon ring of up to 10 atoms in each ring,wherein at least one ring is aromatic, and may be unsubstituted orsubstituted. Examples of such aryl elements include phenyl,p-toluenyl(4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl,biphenyl, phenanthryl, anthryl or acenaphthyl. In cases where the arylsubstituent is bicyclic and one ring is non-aromatic, it is understoodthat attachment is via the aromatic ring. Where an aryl group ispolycyclic, at least 2 aromatic rings are adjacent, ie. share one side.For example, polycyclic aryl groups do not include moieties containing atetracycline structure.

Further, the use of the term “polycyclic” is not limited to aryl groups.The term “polycyclic” as used herein may also refer to unsaturated orpartially unsaturated multiple fused ring structures. However, the term“polycyclic” as used herein in any context excludes the tetracyclinestructure.

The term “arylalkyl” refers to alkyl groups as described above whereinone or more bonds to hydrogen contained therein are replaced by a bondto an aryl group as described above. It is understood that an“arylalkyl” group is connected to a core molecule through a bond fromthe alkyl group and that the aryl group acts as a substituent on thealkyl group. Examples of arylalkyl moieties include, but are not limitedto, benzyl(phenylmethyl),p-trifluoromethylbenzyl(4-trifluoromethylphenylmethyl), 1-phenylethyl,2-phenylethyl, 3-phenylpropyl, 2-phenylpropyl and the like.

The term “heteroaryl”, as used herein, represents a stable monocyclic,bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein atleast one ring is aromatic and contains from 1 to 4 heteroatoms selectedfrom the group consisting of O, N and S. Bicyclic aromatic heteroarylgroups include phenyl, pyridine, pyrimidine or pyridizine rings that are(a) fused to a 6-membered aromatic (unsaturated) heterocyclic ringhaving one nitrogen atom; (b) fused to a 5- or 6-membered aromatic(unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused toa 5-membered aromatic (unsaturated) heterocyclic ring having onenitrogen atom together with either one oxygen or one sulfur atom; or (d)fused to a 5-membered aromatic (unsaturated) heterocyclic ring havingone heteroatom selected from O, N or S. Heteroaryl groups within thescope of this definition include but are not limited to:benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl,benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl,cinnolinyl, furanyl, indolinyl, indolyl, indolazinyl, indazolyl,isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl,naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline,oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl,pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl,quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl,thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl,hexahydroazepinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl,dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl,carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl,benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl,furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl,oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl,pyrimidinyl, pyrrolyl, tetra-hydroquinoline. In cases where theheteroaryl substituent is bicyclic and one ring is non-aromatic orcontains no heteroatoms, it is understood that attachment is via thearomatic ring or via the heteroatom containing ring, respectively. Ifthe heteroaryl contains nitrogen atoms, it is understood that thecorresponding N-oxides thereof are also encompassed by this definition.

The term “heterocycle” or “heterocyclyl” refers to a mono- orpoly-cyclic ring system which can be saturated or contains one or moredegrees of unsaturation and contains one or more heteroatoms. Preferredheteroatoms include N, O, and/or S, including N-oxides, sulfur oxides,and dioxides. Preferably the ring is three to ten-membered and is eithersaturated or has one or more degrees of unsaturation. The heterocyclemay be unsubstituted or substituted, with multiple degrees ofsubstitution being allowed. Such rings may be optionally fused to one ormore of another “heterocyclic” ring(s), heteroaryl ring(s), arylring(s), or cycloalkyl ring(s). Examples of heterocycles include, butare not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane,piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine,tetrahydrothiopyran, tetrahydrothiophene, 1,3-oxathiolane, and the like.

The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclylsubstituents may be substituted or unsubstituted, unless specificallydefined otherwise.

In the compounds of the present invention, alkyl, alkenyl, alkynyl,aryl, heterocyclyl and heteroaryl groups can be further substituted byreplacing one or more hydrogen atoms with alternative non-hydrogengroups. These include, but are not limited to, halo, hydroxy, mercapto,amino, carboxy, cyano and carbamoyl.

As used herein, the term “halogen” refers to F, Cl, Br, and I.

The term “substituted” refers to a functional group as described abovein which one or more bonds to a hydrogen atom contained therein arereplaced by a bond to non-hydrogen or non-carbon atoms, provided thatnormal valencies are maintained and that the substitution results in astable compound. Substituted groups also include groups in which one ormore bonds to a carbon(s) or hydrogen(s) atom are replaced by one ormore bonds, including double or triple bonds, to a heteroatom. Examplesof substituents include the functional groups described above, and, inparticular, halogens (ie., F, Cl, Br, and I); alkyl groups, such asmethyl, ethyl, n-propyl, isopropryl, n-butyl, tert-butyl, andtrifluoromethyl; hydroxyl; alkoxy groups, such as methoxy, ethoxy,n-propoxy, and isopropoxy; aryloxy groups, such as phenoxy;arylalkyloxy, such as benzyloxy (phenylmethoxy) andp-trifluoromethylbenzyloxy(4-trifluoromethylphenylmethoxy);heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl,methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto;sulfanyl groups, such as methylsulfanyl, ethylsulfanyl andpropylsulfanyl; cyano; amino groups, such as amino, methylamino,dimethylamino, ethylamino, and diethylamino; and carboxyl. Wheremultiple substituent moieties are disclosed or claimed, the substitutedcompound can be independently substituted by one or more of thedisclosed or claimed substituent moieties, singly or plurally. Byindependently substituted, it is meant that the (two or more)substituents can be the same or different.

It is understood that substituents and substitution patterns on thecompounds of the instant invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art, as wellas those methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

As used herein, abbreviations are defined as follows:

DNA=deoxyribonucleic acid

RNA=ribonucleic acid

IL=interleukin

MCP=monocyte chemoattractant protein

TNF=tumor necrosis factor

VEGF=vascular endothelial growth factor

MMP=matrix metalloproteinase

LBS=lipopolysaccharide

HPLC=high-performance liquid chromatography

DLAR=Division of Laboratory Animal Resources

In choosing the compounds of the present invention, one of ordinaryskill in the art will recognize that the various substituents, ie. R₁,R₂, etc. are to be chosen in conformity with well-known principles ofchemical structure connectivity.

The various R groups attached to the aromatic rings of the compoundsdisclosed herein may be added to the rings by standard procedures, forexample those set forth in Advanced Organic Chemistry: Part B: Reactionand Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed.Edition. (2007), the content of which is hereby incorporated byreference.

While curcumin has been known to bind metal ions such as those ofcopper, iron, and zinc, affinity for zinc has been shown to be weak(19).

In the subject invention, the improved biological activity of curcuminand its analogues is attributed in part to their ability to access andbind zinc ions and an enhanced solubility. This invention describes thatthe enhancement of zinc binding affinity through the installation ofelectron-withdrawing and electron-donating groups at strategiclocations, namely the C-4 carbon and the aryl rings, on the curcuminskeleton and results in the enhancement of biological activity,including inhibition of MMP activity, NFκB activation, and cytokineproduction.

Without wishing to be bound by theory, it is believed that zinc bindingaffinity arises from increased stability of the curcumin enolate formedby removal of hydrogen from the C-4 carbon, which then proceeds to forma complex with zinc. The stability of a carbanion, including an enolate,is directly related to the acidity of the ionizable hydrogen, such as anenolic hydrogen. In general, the stability of an enolate increases withincreasing acidity of the enolic hydrogen. Herein, the enolic hydrogenrefers to the hydrogen atom connected to the C-4 carbon of the curcuminskeleton.

The acidity of the enolic hydrogen of curcumin and its analogues can beenhanced by incorporation of an electron-withdrawing group at the C-4carbon. Substituents which delocalize negative charge will enhanceacidity and stability of the resulting carbanion, such as an enolate.Again, without wishing to be bound by theory, it is believed that theelectron-withdrawing group allows the negative charge of the enolate tobe delocalized into the electron-withdrawing group, thereby stabilizingthe enolate, enhancing its stability, and increasing its zinc bindingaffinity.

The electronic characteristics of the aryl rings of curcumin are alsorelevant for enhancing zinc binding affinity and biological activity.While not required, electron-donating groups on the aryl portions of thecurcumin skeleton seem to improve its activity. The incorporation ofsuch electron-donating groups on the aryl rings may affect one or morefactors, including enhancement of water solubility and improvement ofcation-pi interactions. Without wishing to be bound by theory, theinstallation of electron-donating groups on the aryl rings, inconjunction with the choice of C-4 electron-withdrawing group, isbelieved to increase electron polarization within the molecule such thatintermolecular dipole-dipole forces with surrounding water molecules isenhanced, thereby increasing water solubility. Electron-donating groupsmay also increase water solubility by enhancing hydrogen-bondinginteractions with surrounding water molecules. Furthermore, with respectto cation-pi interactions, it is believed that electron-donating groupsincrease electron density on the aryl rings, thereby enhancing thearyls' ability to recognize and/or bind to cations or cation-containingproteins.

The choice of electron-withdrawing groups on the C-4 carbon and thechoice of electron-donating groups on the aryl rings may be chosen usingtechniques well known by the ordinarily skilled artisan. In general, theelectron donating ability of common substituents suitable for use on thearyl rings can be estimated by their Hammett σ values. The Hammettσ_(para) value is a relative measurement comparing the electronicinfluence of the substituent in the para position of a phenyl ring tothe electronic influence of a hydrogen substituted at the para position.Typically for aromatic substituents in general, a negative Hammettσ_(para) value is indicative of a group or substituent having anelectron-donating influence on a pi electron system (ie., anelectron-donating group) and a positive Hammett σ_(para) value isindicative of a group or substituent having an electron-withdrawinginfluence on a pi electron system (ie., an electron-withdrawing group).Similarly, Hammett σ_(meta) value is a relative measurement comparingthe electronic influence of the substituent in the meta position of aphenyl ring to the electronic influence of a hydrogen substituted at themeta position. A list of Hammett σ_(para) and σ_(neta) values for commonsubstituents can be found in Lowry and Richardson, “Mechanism and Theoryin Organic Chemistry”, 3rd ed, p. 144. The effect of some substituents,including some electron-withdrawing groups, on C—H acidity can also befound on page 518 in Lowry and Richardson, “Mechanism and Theory inOrganic Chemistry”, 3rd ed, the content of which is hereby incorporatedby reference.

Computation methods may be used by the ordinarily skilled artisan toquantify and predict the effects of the chosen electron-donating andelectron-withdrawing groups on curcumin zinc-binding affinity, watersolubility and pKa (acid dissociation constant) of ionizable hydrogens,among other pharmaceutical properties. Commercially-available softwarethat may be used, alone or in combination, for such computationalmethods includes, but is not limited to, ACD/PhysChem Suite® (AdvancedChemical Development, Inc., Ontario, Canada), Gaussian 03 (Gaussian,Inc., Wallingford, Conn.), Spartan® (Wavefunction, Inc., Irvine,Calif.), MacroModel and QikProp (Schrödinger, Inc., New York, N.Y.).

As used herein, the term “electron-withdrawing group” refers to asubstituent or functional group that has the property of increasingelectron density around itself relative to groups in its proximity.Electron withdrawing property is a combination of induction andresonance. Electron withdrawal by induction refers to electron clouddisplacement towards the more electronegative of two atoms in a σ-bond.Therefore, the electron cloud between two atoms of differingelectronegativity is not uniform and a permanent state of bondpolarization occurs such that the more electronegative atom has a slightnegative charge and the other atom has a slight positive charge.Electron withdrawal by resonance refers to the ability of substituentsor functional groups to withdraw electron density on the basis ofrelevant resonance structures arising from p-orbital overlap. Suitableelectron-withdrawing groups include, but are not limited to, —CN, —CF₃,halogen, —NO₂, —OCF₃, —OR₁₂, —NHCOR₁₂, —SR₁₂, —SO₂R₁₃, —COR₁₄, —CSR₁₄,—CNR₁₄, —C(═NR₁₂)R₁₄, —C(═NH)R₁₄, —SOR₁₂, —POR₁₂, —P(═O)(OR₁₂)(OR₁₃), or—P(OR₁₂)(OR₁₃),

-   -   wherein R₁₂ and R₁₃ are each, independently, H, C₁₋₁₀ alkyl,        C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;    -   R₁₄ is C₂₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroaryl,        heterocyclyl, methoxy, —OR₁₅, —NR₁₆R₁₇, or

-   -    wherein R₁₅ is H, C₃₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl;        -   R₁₅ and R₁₇ are each, independently, H, C₁₋₁₀ alkyl, C₂₋₁₀            alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;        -   R₁₈, R₁₉, R₂₁, and R₂₂ are each independently H, halogen,            —NO₂, —CN, —NR₂₃R₂₄, —SR₂₃, —SO₂R₂₃, —CO₂R₂₃, —OR₂₅, CF₃,            —SOR₂₃, —POR₂₃, —C(═S)R₂₃, —C(═NH)R₂₃, C(═NR₂₄)R₂₃,            —C(═N)R₂₃, P(═O)(OR₂₃)(OR₂₄)—(OR₂₃)(OR₂₄), —C(═S)R₂₃, C₁₋₁₀            alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or            heterocyclyl;            -   wherein R₂₃, R₂₄, and R₂₅ are each, independently, H,                C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl,                heteroaryl, or heterocyclyl;        -   R₂₀ is halogen, —NO₂, —CN, —NR₂₆R₂₇, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀            alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl;            -   wherein R₂₆ and R₂₇ are each, independently, H, C₁₋₁₀                alkyl, C₂₋₂₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl,                or heterocyclyl.

The compounds of the instant invention may be in a salt form. As usedherein, a “salt” is the salt of the instant compounds which has beenmodified by making acid or base salts of the compounds. Acidicsubstances can form salts with acceptable bases, including, but notlimited to, lysine, arginine, and the like. In the case of compoundsadministered to a subject, e.g. a human, the salt is pharmaceuticallyacceptable. Examples of pharmaceutically acceptable salts include, butare not limited to, mineral or organic acid salts formed at basicresidues such as amino groups; alkali or organic base salts formed atacidic residues such as phenols, carboxylic acids, and carbons having atleast 1 acidic hydrogen atom adjacent to a carbonyl. Where acid saltsare formed, such salts can be made using an organic or inorganic acid.Such acid salts include, but are not limited to, chlorides, bromides,sulfates, nitrates, phosphates, sulfonates, formates, tartrates,maleates, malates, citrates, benzoates, salicylates, ascorbates, and thelike. Because the compounds of the subject invention also possesscarbons having at least 1 acidic hydrogen atom adjacent to a carbonyl,enolate salts may be formed by reaction with a suitable base. Suitablebases include, but are not limited, to inorganic bases, such as alkaliand alkaline earth metal hydroxides; and organic bases, including, butnot limited to, ammonia, alkyl amines, amino alcohols, amino sugars,amino acids, such as glycine, histidine, and lysine, and alkali metalamides, such as lithium diisopropylamide. The term “pharmaceuticallyacceptable salt” in this respect, refers to the relatively non-toxic,inorganic and organic acid or base addition salts of compounds of thepresent invention. These salts can be prepared in situ during the finalisolation and purification of the compounds of the invention, or byseparately reacting a purified compound of the invention in its freebase or free acid form with a suitable organic or inorganic acid orbase, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, e.g., Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

The compounds and compositions of this invention may be administered invarious forms, including those detailed herein. The treatment with thecompound may be a component of a combination therapy or an adjuncttherapy, ie. the subject or patient in need of the drug is treated orgiven another drug for the disease in conjunction with one or more ofthe instant compounds. This combination therapy can be sequentialtherapy where the patient is treated first with one drug and then theother or the two drugs are given simultaneously. These can beadministered independently by the same route or by two or more differentroutes of administration depending on the dosage forms employed.

As used herein, a “pharmaceutically acceptable carrier” is apharmaceutically acceptable solvent, suspending agent or vehicle, fordelivering the instant compounds to the animal or human. The carrier maybe liquid or solid and is selected with the planned manner ofadministration in mind. Liposomes are also a pharmaceutically acceptablecarrier.

The dosage of the compounds administered in treatment will varydepending upon factors such as the pharmacodynamic characteristics of aspecific chemotherapeutic agent and its mode and route ofadministration; the age, sex, metabolic rate, absorptive efficiency,health and weight of the recipient; the nature and extent of thesymptoms; the kind of concurrent treatment being administered; thefrequency of treatment with; and the desired therapeutic effect.

A dosage unit of the compounds may comprise a single compound ormixtures thereof with other compounds also used to treat rheumatoidarthritis (RA), osteoarthritis (OA), metastases, periodontal disease,such as periodontitis, angiogenesis, emphysema, acute respiratorydistress syndrome, multiple sclerosis, cardiovascular disease, such asatherosclerosis, myocardial infarction, arterial restenosis afterangioplasty and aneurysm development; inflammatory disorders, includingneuroinflammation and inflammatory bowel disease; many types of cancer,including breast cancer, skin cancer, including, but not limited to,melanoma, and prostate cancer; diabetes, stroke, peripheral neuropathy,brain trauma, pancreatitis, and skin disorders, including, but notlimited to, wounds, including ulcers of the skin, accelerated aging, andinflammatory diseases of the skin; bone diseases including, but notlimited to, osteoperosis. The compounds can be administered in oraldosage forms as tablets, capsules, pills, powders, granules, elixirs,tinctures, suspensions, syrups, and emulsions. The compounds may also beadministered in intravenous (bolus or infusion), intraperitoneal,subcutaneous, or intramuscular form, or introduced directly, e.g. bytopical administration, injection or other methods, to the afflictedarea, such as a wound, including ulcers of the skin, all using dosageforms well known to those of ordinary skill in the pharmaceutical arts.

The compounds can be administered in admixture with suitablepharmaceutical diluents, extenders, excipients, or carriers(collectively referred to herein as a pharmaceutically acceptablecarrier) suitably selected with respect to the intended form ofadministration and as consistent with conventional pharmaceuticalpractices. The unit will be in a form suitable for oral, rectal,topical, intravenous or direct injection or parenteral administration.The compounds can be administered alone but are generally mixed with apharmaceutically acceptable carrier. This carrier can be a solid orliquid, and the type of carrier is generally chosen based on the type ofadministration being used. In one embodiment the carrier can be amonoclonal antibody. The active agent can be co-administered in the formof a tablet or capsule, liposome, as an agglomerated powder or in aliquid form. Examples of suitable solid carriers include lactose,sucrose, gelatin and agar. Capsule or tablets can be easily formulatedand can be made easy to swallow or chew; other solid forms includegranules, and bulk powders. Tablets may contain suitable binders,lubricants, diluents, disintegrating agents, coloring agents, flavoringagents, flow-inducing agents, and melting agents. Examples of suitableliquid dosage forms include solutions or suspensions in water,pharmaceutically acceptable fats and oils, alcohols or other organicsolvents, including esters, emulsions, syrups or elixirs, suspensions,solutions and/or suspensions reconstituted from non-effervescentgranules and effervescent preparations reconstituted from effervescentgranules. Such liquid dosage forms may contain, for example, suitablesolvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, thickeners, and melting agents. Oral dosage formsoptionally contain flavorants and coloring agents. Parenteral andintravenous forms may also include minerals and other materials to makethem compatible with the type of injection or delivery system chosen.

Specific examples of pharmaceutical acceptable carriers and excipientsthat may be used to formulate oral dosage forms of the present inventionare described in U.S. Pat. No. 3,903,297 to Robert, issued Sep. 2, 1975.Techniques and compositions for making dosage forms useful in thepresent invention are described-in the following references: 7 ModernPharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979);Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel,Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976);Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company,Easton, Pa., 1985); Advances in Pharmaceutical Sciences (DavidGanderton, Trevor Jones, Eds., 1992); Advances in PharmaceuticalSciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds.,1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugsand the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989);Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs andthe Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); DrugDelivery to the Gastrointestinal Tract (Ellis Horwood Books in theBiological Sciences. Series in Pharmaceutical Technology; J. G. Hardy,S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and thePharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T.Rhodes, Eds.). All of the aforementioned publications are incorporatedby reference herein.

Tablets may contain suitable binders, lubricants, disintegrating agents,coloring agents, flavoring agents, flow-inducing agents, and meltingagents. For instance, for oral administration in the dosage unit form ofa tablet or capsule, the active drug component can be combined with anoral, non-toxic, pharmaceutically acceptable, inert carrier such aslactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose,magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol,sorbitol and the like. Suitable binders include starch, gelatin, naturalsugars such as glucose or beta-lactose, corn sweeteners, natural andsynthetic gums such as acacia, tragacanth, or sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes, and the like.Lubricants used in these dosage forms include sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate, sodiumchloride, and the like. Disintegrators include, without limitation,starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

The compounds can also be administered in the form of liposome deliverysystems, such as small unilamellar vesicles, large unilamallar vesicles,and multilamellar vesicles. Liposomes can be formed from a variety ofphospholipids, such as cholesterol, stearylamine, orphosphatidylcholines. The compounds may be administered as components oftissue-targeted emulsions.

The compounds may also be coupled to soluble polymers as targetable drugcarriers or as a prodrug. Such polymers include polyvinylpyrrolidone,pyran copolymer, polyhydroxylpropylmethacrylamide-phenol,polyhydroxyethylasparta-midephenol, or polyethylene oxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds may becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polyglycolicacid, copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacylates, and crosslinked or amphipathicblock copolymers of hydrogels.

The term “prodrug” as used herein refers to any compound that whenadministered to a biological system generates the compound of theinvention, as a result of spontaneous chemical reaction(s), enzymecatalyzed chemical reaction(s), photolysis, and/or metabolic chemicalreaction(s). A prodrug is thus a covalently modified analog or latentform of a compound of the invention.

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets, powders, and chewing gum; or in liquid dosageforms, such as elixirs, syrups, and suspensions, including, but notlimited to, mouthwash and toothpaste. It can also be administeredparentally, in sterile liquid dosage forms.

Solid dosage forms, such as capsules and tablets, may be enteric coatedto prevent release of the active ingredient compounds before they reachthe small intestine. Materials that may be used as enteric coatingsinclude, but are not limited to, sugars, fatty acids, waxes, shellac,cellulose acetate phthalate (CAP), methyl acrylate-methacrylic acidcopolymers, cellulose acetate succinate, hydroxy propyl methyl cellulosephthalate, hydroxy propyl methyl cellulose acetate succinate(hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP),and methyl methacrylate-methacrylic acid copolymers.

Gelatin capsules may contain the active ingredient compounds andpowdered carriers, such as lactose, starch, cellulose derivatives,magnesium stearate, stearic acid, and the like. Similar diluents can beused to make compressed tablets. Both tablets and capsules can bemanufactured as immediate release products or as sustained releaseproducts to provide for continuous release of medication over a periodof hours. Compressed tablets can be sugar coated or film coated to maskany unpleasant taste and protect the tablet from the atmosphere, orenteric coated for selective disintegration in the gastrointestinaltract.

For oral administration in liquid dosage form, the oral drug componentsare combined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Examples ofsuitable liquid dosage forms include solutions or suspensions in water,pharmaceutically acceptable fats and oils, alcohols or other organicsolvents, including esters, emulsions, syrups or elixirs, suspensions,solutions and/or suspensions reconstituted from non-effervescentgranules and effervescent preparations reconstituted from effervescentgranules. Such liquid dosage forms may contain, for example, suitablesolvents, preservatives, emulsifying agents, suspending agents,diluents, sweeteners, thickeners, and melting agents.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance. In general, water, a suitableoil, saline, aqueous dextrose (glucose), and related sugar solutions andglycols such as propylene glycol or polyethylene glycols are suitablecarriers for parenteral solutions. Solutions for parenteraladministration preferably contain a water soluble salt of the activeingredient, suitable stabilizing agents, and if necessary, buffersubstances. Sustained release liquid dosage forms suitable forparenteral administration, including, but not limited to, water-in-oiland oil-in-water microemulsions and biodegradable microsphere polymers,may be used according to methods well-known to those having ordinaryskill in the art. Antioxidizing agents such as sodium bisulfite, sodiumsulfite, or ascorbic acid, either alone or combined, are suitablestabilizing agents. Also used are citric acid and its salts and sodiumEDTA. In addition, parenteral solutions can contain preservatives, suchas benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field. Solubilizing agents may be used to enhancesolubility of the compounds of the subject invention in the liquiddosage form. Suitable solubilizing agents include, but are not limitedto, amines, amino alcohols, amino sugars, and amino acids, such asglycine, histidine, and lysine.

The compounds of the instant invention may also be administered inintranasal form via use of suitable intranasal vehicles, or viatransdermal routes, using those forms of transdermal skin patches wellknown to those of ordinary skill in that art. To be administered in theform of a transdermal delivery system, the dosage administration willgenerally be continuous rather than intermittent throughout the dosageregimen.

Parenteral and intravenous forms may also include minerals and othermaterials to make them compatible with the type of injection or deliverysystem chosen.

The compounds and compositions of the invention can be coated ontostents for temporary or permanent implantation into the cardiovascularsystem of a subject.

The compounds and compositions of the subject invention, like curcuminand other curcumin analogues, are useful for treating rheumatoidarthritis (RA), osteoarthritis

(OA), metastases, periodontal disease, such as periodontitis,angiogenesis, emphysema, acute respiratory distress syndrome, multiplesclerosis, cardiovascular disease, such as atherosclerosis, myocardialinfarction, arterial restenosis after angioplasty and aneurysmdevelopment; inflammatory disorders, including neuroinflammation andinflammatory bowel disease; many types of cancer, including breastcancer, skin cancer, including, but not limited to, melanoma, andprostate cancer; diabetes, stroke, peripheral neuropathy, brain trauma,and pancreatitis; bone diseases including, but not limited to,osteoperosis.

Curcumin has been known to be useful in the treatment of skin disorders,including, but not limited to, wounds, psoriasis, acne, burns, eczema,as well as inflammation accompanying such disorders (20-24). Singer andco-workers have shown that curcumin reduces burn progression in rats(21) and Sidhu and co-workers have shown curcumin to be effective inenhancing wound healing in animals (22), includingstreptozoticin-induced diabetic rats and genetically diabetic mice (23).In addition, Phan and co-workers have shown that curcumin exhibitspowerful inhibition against hydrogen peroxide damage in humankeratinocytes and fibroblasts (24). Accordingly, the improved compoundsand compositions of the subject invention are useful for the treatmentof skin disorders, including, but not limited to, wounds, includingulcers of the skin, and inflammatory diseases of the skin.

Variations on the following general synthetic methods will be readilyapparent to those skilled in the art and are deemed to be within thescope of the present invention (47).

The synthesis of the curcumin analogues of the present invention can becarried out according to general scheme 1. The R groups designate anynumber of generic substituents.

The starting material is provided by 2,4-pentanedione, which issubstituted at the 3-carbon (see compound a). The desired substituted2,4-pentanedione may be purchased from commercial sources or it may besynthesized using conventional functional group transformationswell-known in the chemical arts, for example, those set forth in OrganicSynthesis, Michael B. Smith, (McGraw-Hill) Second ed. (2001) and March'sAdvanced Organic Chemistry: Reactions, Mechanisms, and Structure,Michael B. Smith and Jerry March, (Wiley) Sixth ed. (2007), andspecifically by Bingham and Tyman (45) and in the case of3-aryl-aminocarbonyl compounds by Dieckman, Hoppe and Stein (46), thecontents of which are hereby incorporated by reference. 2,4-pentanedionea is reacted with boron trioxide to form boron enolate complex b.

Boron enolate complex b is a complex formed by coordination of theenolate of compound a with boron. It is understood by those havingordinary skill in the art that the number of compound a enolates thatmay coordinate to boron as well as the coordination mode, ie.monodentate versus bidentate, are variable so long as reaction, such asKnoevenagel condensation, at the C-3 carbon of the 2,4-pentanedione issuppressed.

Boron enolate complex b is then exposed to a benzaldehyde compound inthe presence of a base catalyst and a water scavenger to form curcuminanalogue c via aldol condensation. The ordinarily skilled artisan willappreciate that the benzaldehyde may possess various substituents on thephenyl ring so long as reactivity at the aldehyde position is nothindered. Substituted benzaldehyde compounds may be purchased fromcommercial sources or readily synthesized using aryl substitutionchemistry that is well-known in the art. Suitable base catalysts for thealdol step include, but are not limited to, secondary amines, such asn-butylamine and n-butylamine acetate, and tertiary amines. Suitablewater scavengers include, but are not limited to, alkyl borates, such astrimethyl borate, alkyl phosphates, and mixtures thereof. Other suitablereaction parameters have also been described by Krackov and Bellis inU.S. Pat. No. 5,679,864, the content of which is hereby incorporated byreference.

All combinations of the various elements described herein are within thescope of the invention.

Herein, where chemical substituents are disclosed in the alternative, itis intended that each such substituent can be used or combined with oneor more other substituents disclosed in the alternative.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS

General Procedure for the Synthesis of Chemically-Modified Curcumins:

Boron oxide (0.49 g, 7 mmol, 0.7 eq.) and3-methoxycarbonyl-pentane-2,4-dione (45) (1.58 g, 10 mmol, 1.0 eq.) forin the cases of compounds 11, 12 and 13, 10 mmol. of3-(N-phenylaminocarbonyl)pentane-2,4-dione (46)} were placed in a 50 mLflask and the mixture was heated to 90° C. for 5 min to form apale-yellow glass. The selected aromatic aldehyde (20 mmol, 2.0 eq.) andtrimethyl borate (4.16 g, 40 mmol, 4.0 eq.) were dissolved in dry ethylacetate (10 mL) and gradually added to the flask (3 min). with magneticstirring. Then butylamine (0.05 mL) and butylammonium acetate indimethylformamide solution (0.2 mL; 0.136 g/mL) were added. After about1 hour, a precipitate began to form and stirring was continued at roomtemperature for 48 hours. The reddish precipitate was removed byfiltration, washed with diethyl ether (5 mL). then dissolved in methanol(50 mL) and boiled for 30 min during which time the color became muchlighter. Methanol was then removed by rotary-evaporation and the crudeproduct was purified by crystallization from dichloromethane (20 mL) andmethanol (20 mL).

Example 1 Preparation of1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione(1)

A mixture of boron trioxide (B₂O₃: 1.5 g) and3-methoxycarbonylpentan-2,4-dione (2.5 g) were fused at 120° C. until ahomogeneous melt was formed (3 min). 3-methoxycarbonylpentan-2,4-dionecan be synthesized using known methods (25, 26). For example, reactionof 3-acetylthiotetronic acid, which is synthesized by a known method(25), with methanol yields 3-methoxycarbonylpentan-2,4-dione (26). Tothe homogenous melt, there was then added a solution of trimethyl borate(6.0 g) and vanillin (4.5 g) in ethyl acetate (15 mL) followed bybutylamine acetate (0.068 g) in dimethylformamide solution (0.5 mL)followed by 2 drops (Pasteur pipet) of butylamine. The mixture, whenstirred, quickly became homogeneous and after three hours began to forma red precipitate, which was complete after standing for 24 h. Thesolution was filtered and the solid (7.5 g) that was collected waswashed with ether, dried, and added to methanol (60 mL). This mixturewas boiled gently for three hours with slow distillation thenconcentrated to 30 mL when the product, compound 2, (4.4 g) crystallizedspontaneously. Melting point (mp): 178-179° C. Concentration of themother liquors gave a second crop (0.3 g) of identical mp. Total yield,73.6% based on vanillin. Mass spectrum m/e: Found 426.01; Calcd. 426.25.1H NMR CDCl₃: Significant peaks δ 3.944 (s. 3H, OCH ₃), 3.952 (s, 6H OCH₃), 5.89 (s, 2H, OH) 18.3 (S, 1H, H at C4). Aromatic and ethylenicprotons 6.9-7.3 (12H) as expected.

Chemically-modified curcumins possessing an electron-withdrawing groupat the C-4 carbon are demonstrated to have improved inhibition of MMPactivity, inhibition of NFκB activation, and inhibition ofpro-inflammatory cytokine production.

The following compounds were made according to the general proceduredescribed hereinabove.

1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione(1): 72.0% yield. mp 175-176° C. Mass spectrum m/e 425.1 (M−1)⁺; Calcd.426.0. ¹H NMR (CDCl₃): Significant peaks 3.880 (s. 6H, aromatic OCH₃)3.760 (s. 3H, ester OCH₃), 17.960 (s. 1H, H at C4). Aromatic andethylenic protons 7.3-8.8 (10H).

1,7-Bis(4-hydroxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione(3): 49.2% yield. mp 214-216° C. Mass spectrum m/e: 365.0 (M−1)⁺; Calcd.365.1. ¹H NMR (CDCl₃): Significant peaks 3.777 (s. 3H, OCH₃), 18.164 (s.1H, H at C4), 9.320 (s. 2H, OH). Aromatic and ethylenic protons 6.8-7.7(12H).

1,7-Bis(4-hydroxy-3-methoxyphenyl)-4-N-phenylaminocarbonylhepta-1E,6E-dien-3,5-dione(6): 11.0% yield. mp 193-194° C. Mass spectrum m/e: 486.2 (M−1)⁺; Calcd.486.1. ¹H NMR (DMSO-d₆): Significant peaks 17.570 (s. 1H, H at C4),10.580 (s. H, NH), 9.777 (s. 2H, OH), 3.701 (s. 6H, OCH₃). Aromatic andethylenic protons 6.7-7.8 (10H).

1,7-Bis(4-hydroxyphenyl)-4-N-phenylaminocarbonylhepta-1E,6E-dien-3,5-dione(7): 10.2% yield. mp 220-221° C. Mass spectrum m/e: 426.2 (M−1)⁺; Calcd.426.1. ¹H NMR (DMSO-d₆): Significant peaks 17.561 (s. 1H, H at C4),10.609 (s. H, NH), 10.159 (s. 2H, OH). Aromatic and ethylenic protons6.7-7.8 (12H).

1,7-Bis(4-dimethylaminophenyl)-4-N-phenylaminocarbonylhepta-1E,6E-dien-3,5-dione(8): 26.8% yield. mp 208-209° C. Mass spectrum m/e: 480.3 (M−1)⁺; Calcd.480.4. ¹H NMR (DMSO-d₆): Significant peaks 17.773 (s. 1H, H at C4),10.556 (s. H, NH), 2.959 (s. 12H, N(CH₃)₂). Aromatic and ethylenicprotons 6.7-7.8 (12H).

1,7-Bis(3-pyridyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione (9):38.7% yield mp. 195-196′C. Mass spectrum m/e: 335.69 (M−1)⁺; Calcd.335.01. ¹H NMR (CDCl₃): Significant peaks 3.960 (s. 3H, OCH₃), 18.050(s. 1H, H at C4). Aromatic and ethylenic protons 7.0-8.6 (12H).

1,7-Bis(2-hydroxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione(10): 46.3% yield. mp 165-166° C. Mass spectrum m/e: 365.1 (M−1)⁺;Calcd. 365.1. ¹H NMR (CDCl₃): Significant peaks 3.610 (s. 3H, OCH₃),17.980 (s. 1H, H at C4), 9.420 (s. 2H, OH). Aromatic and ethylenicprotons 6.4-7.9 (12H).

1,7-Bis(3-hydroxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione(11): 20.2% yield. mp 188-189° C. Mass spectrum m/e: 365.1 (M−1)⁺;Calcd. 365.1. ¹H NMR (CDCl₃): Significant peaks 3.851 (s. 3H, OCH₃),18.010 (s. 1H, H at C4), 8.890 (s. 2H, OH). Aromatic and ethylenicprotons 6.7-7.7 (12H).

1,7-Bis(3-nitro-4-hydroxy-5-methoxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione(12): 26.0% yield. mp N/A. Mass spectrum m/e: 515.2 (M−1)⁺; Calcd.515.1. ¹H NMR (CDCl₃): Significant peaks 3.873 (s. 3H, OCH₃), 18.056 (s.1H, H at C4), 11.010 (s. 2H, OH). Aromatic and ethylenic protons 7.3-8.8(8H).

1,7-Bis(4-hydroxy-3,5-dimethoxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione(13): 77.0% yield. mp 179-180° C. Mass spectrum m/e: 485.2 (M−1)⁺;Calcd. 485.0. ¹H NMR CDCl₃: Significant peaks 3.925 (s. 3H, ester OCH₃),3.948 (s. 12H, aromatic OCH₃), 18.336 (s. 1H, H at C4). Aromatic andethylenic protons 6.8-7.8 (8H).

1,7-Bis(4-N,N-dimethylaminophenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione(14): 45.1% yield. mp N/A. Mass spectrum m/e: 421.4 (M+1)⁺; Calcd.421.1. ¹H NMR (CDCl₃): Significant peaks 3.938 (s. 3H, OCH₃), 18.486(3.1H, H at C4), 3.028 (s. 12H, N(CH₃)₂). Aromatic and ethylenic protons6.6-7.9 (12H).

1,7-Bis(2-hydroxy-3-methoxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione(15): 25.8% mp 201-202° C. Mass spectrum m/e: 427.4 (M+1)⁺; Calcd.426.0. ¹H NMR (CDCl₃): Significant peaks 3.824 (s. 6H, aromatic OCH₃)3.878 (s. 3H, ester OCH₃), 18.125 (s. 1H, H at C4), 9.609 (s. 2H, OH).Aromatic and ethylenic protons 6.8-8.1 (10H).

1,7-Bis(4-acetoxy-3-methoxyphenyl)-4-methoxycarbonylhepta-1E,6E-dien-3,5-dione(16): 46.0% mp 169-170° C. Mass spectrum m/e: 510.1 (M−1)⁺; Calcd.510.1. ¹H NMR (CDCl₃): Significant peaks 3.610 (s. 3H, ester OCH₃),3.869 (s. 6H, aromatic OCH₃), 18.175 (s. 1H, H at C4). Aromatic andethylenic protons 7.0-7.9 (12H).

Mass spectral data are reported in negative- or positive-ion modedepending on the specific compound.

Example 2

Inhibition of MMPs

It has been observed that 50 and 100 μM concentrations of curcumindecreased TNFα production by endotoxin-stimulated human monocytes (HMs)in culture by 80-90% (lower concentrations of curcumin, 10 and 20 μM,had no effect). However, this inhibitory effect was associated with someprecipitation of the curcumin in cell culture and with significantcytotoxicity. It was hypothesized that increasing the solubility ofcurcumin will: (i) enhance its efficacy as an inhibitor of cytokineexpression, (ii) reduce its cytotoxicity, and (iii) preserve (perhapseven enhance; see below) its potency, as an MMP inhibitor (MMPI)compound, which was found to be similar to that of the Zn⁺⁺ chelatingcompound, 1,10-O-phenanthroline (FIG. 1). However, it should be notedthat excessive inhibition of MMP activity may not be desirabletherapeutically because a minimal, or basal, level of MMPs may benecessary for optimal defense of the host (27).

Two chemically-modified curcumins, compound 1 and 2, were generated withimproved solubility and tested in vitro for their proteinase inhibitorpotency (IC₅₀) and in a cell culture system for cytotoxicity andanti-inflammatory properties.

Table 1 shows the IC₅₀ of curcumin, compound 1, and compound 2, comparedto a standard Zn⁺⁺ binding & MMPI (matrix metalloproteinase inhibitor),1,10-O-phenanthroline (o-phen), against purified human PMN MMP-8 (fromEMD biologics, Inc., Gibbstown, N.J.) using a synthetic octapeptidecontaining the collagenase-susceptible glycine-isoleucine peptide bondand measuring the tripeptide breakdown products by HPLC (Waters Alliance2695 System with a reverse phase C-18 column). Compound 1 was anexcellent MMPI with an IC₅₀ equivalent to that of 1,10-O-phenanthroline,while compound 2, which lacked substituents on the aryl moieties, didnot show a dose response.

TABLE 1 MMP inhibition in vitro Ratio of each IC₅₀ against IC₅₀ to IC₅₀of purified 1,10-O- Compound hMMP-8 phenanthroline

10-35 μM* 1 = 35 μM 1 = 10 μM

14 μM 1.4

35 μM 1

No dose response —

28 μM 0.8 *in one experiment, the IC₅₀ for phenanthroline was 10 μM, ina second experiment the IC₅₀ for phenanthroline was 35 μM.

Example 3

The cytotoxicity of curcumin and compound 1 was compared to that of1,10-O-phenanthroline in human monocytes. The results are summarized intable 2.

TABLE 2 Cytotoxicity in human monocytes Recovery CytotoxicityCytotoxicity % Inhibition from Monocytes Monocytes of MMP-9 in human @ 5h @ 18 h monocyte Compound serum incubation incubation media 1,10-O-phe-— — — — nanthroline curcumin 83-94% 10 uM- 0%  10 uM- 0%  nd 20 uM- 20%20 uM- 20% 50 uM- 50% 50 uM- 77% compound 1 76-80% 2 uM- 0% 2 uM- 0%  2uM +++++ 10 uM- 0%    25 uM- 45.5% 25 uM +++++ 25 uM- 33% 50 uM- 53% 50uM +++++

Detection of compounds & extractability/recovery from human serum (forfuture use to determine in vivo pharmacokinetics) was achieved usingHPLC. The HPLC detection method is briefly described herein. 50 μl ofbuffer or human serum containing 166.67 μM, 16.67 μM and 1.67 μM of thecompounds were incubated with 100 μl pre-cooled (−10° C.) extractionsolvent containing CAN-MeOH-0.5M oxalic acid at a ratio of 60:30:10. Themixture was vortexed and centrifuged at 10,000 rpm for 10 min. Thesupernatant was then aliquoted and injected into an HPLC for analysis.Stock solutions of the compounds were made 100× in DMSO, then furtherdiluted with buffer or serum. Results show 83-94% recovery for curcumin,and 76-80% recovery for compound 1 (see table 2).

Example 4

Inhibition of Pro-Inflammatory Cytokine Production

Inhibition of pro-inflammatory cytokine production by compound 1 wasexamined. Briefly, human peripheral blood monocytes (HMs) were isolatedfrom a leukocyte concentrate by density gradient centrifugation(Lymphoprep) and the isolated HMs cultured (2 h, 37° C.) to removenon-adherent cells. The adherent HMs were cultured for 18 h (1×10⁶cells/well, 24-well plates)±endotoxin (LPS)±different compoundsdissolved in DMSO (the final concentration of DMSO in the RPMIserum-free culture media with Pen./Strep. antibiotics was no greaterthan 0.5%). After incubation, the proinflammatory cytokines, TNFα,IL-1β, MCP-1 and IL-6 were measured in the conditioned media by ELISAand cytotoxicity was assessed by measuring the absorbance (490 nm) offormazan produced by reduction of MTS. The HMs alone ±0.1-0.5% DMSOproduced minimal levels of these pro-inflammatory cytokines. However,addition of 100 ng/ml or 10 μg/ml of LPS both dramatically stimulatedcytokine production. Of the tested compounds, compound 1 stood out ashaving improved inhibition of proinflammatory cytokines when compared tocurcumin. At 2 μM concentration, compound 1 showed no evidence ofcytotoxicity (as shown on table 2), but inhibited cytokine production asfollows: TNFα, IL-1β, MCP-1 and IL-6 were inhibited by 63%, 41%, 74% and30% respectively (see table 3).

TABLE 3 Inhibition of cytokine production % Inhibition of % Inhibitionof % Inhibition of % Inhibition of TNF-α produced by IL-1β produced byMCP-1 produced by IL-6 produced by stimulated Monocytes stimulatedMonocytes stimulated Monocytes stimulated Monocytes Compound with LPS @18 h with LPS @ 18 h with LPS @ 18 h with LPS @ 18 h1,10-O-phenanthroline — — — — curcumin 10 uM- 0% nd nd nd 20 uM- 0%  50uM- 80% compound 1  2 uM- 63% 2 uM- 41%  2 uM- 74% 2 uM- 30%  25 uM-100% 25 uM- 100% 25 uM- 78% 25 uM- 100%  50 uM- 100% 50 uM- 100% 50 uM-79% 50 uM- 100%

The cell culture experiment was repeated to confirm the improvedproinflammatory cytokine inhibition activity of compound 1. FIG. 2 showsthat compound 1 at 2 μM concentration was indeed potent in reducingproinflammatory cytokines such as MCP-1 and TNFα and growth factors suchas VEGF in LPS-challenged human monocytes in culture. The cytokinelevels were measured by Luminex multiplex, a method that allows thesimultaneous measurement of different biological markers.

Example 5

Inhibition of NFκB Activation in Human Monocytes

The inhibition of NFκB activation by compound 1 was studied in humanmonocytes in cell culture. Compound 1 was examined at 2 μM and 10 μMconcentration in the presence of 2 different activation stimuli, LPSendotoxin and CRP/oxid-LDL complex. It was observed that compound 1inhibited both endotoxin-stimulated NFκB activation and CRP/oxid-LDLcomplex-stimulated NFκB activation (see table 4). Preliminary studiesindicated that compound 1 had little/no effect on p38 MAP kinaseactivation.

TABLE 4 The effect of compound 1 on NFκB activation in human monocytesin cell culture. Activation stimulus Concentration % InhibitionEndotoxin (LPS)  2 μM 24 10 μM 85 CRP/oxid-LDL  2 μM 91 Complex 10 μM100

Example 6

Development of a Rat Model of Type 1 Diabetes

A rat model of type I diabetes was developed to test the efficacy of thecompounds of the subject invention in vivo. This animal model ischaracterized, in part, by excessive MMP activity, collagen breakdown,and proinflammatory cytokine expression. In a previous drug developmentprogram, resulting in two FDA-approved systemically-administered (by theoral route) drugs currently on the market (in the U.S. and Europe forone, and in the U.S., Canada, and Europe for the other), this rat modelproved very effective for testing efficacy and pharmacokinetics (e.g.,serum half-life) in vivo and produced results consistent with those fromour in vitro and cell culture studies (4, 28, 29).

A well-established model of diabetes induction in Sprague-Dawley ratswith and without the additional induction of periodontal disease is tobe utilized to investigate the mechanism underlying the associationbetween diabetes and bone loss in periodontal disease (30-41).

DLAR was used for both housing and experimental manipulations to allowclose monitoring of treated animals and provide immediate access toanalgesia or euthanasia as required. Rats were allowed to aclimate tothe facility for at least three days prior to experimental use. Alllaboratory and animal care personnel were trained in the propermanipulation and care of rats.

Investigational focus includes three general areas: (I) the effect ofmatrix metalloproteinases (MMPs) in the tissue destruction and immuneresponses related to inflammation, (II) the effect of prophylacticand/or therapeutic treatment by administration of the compounds onmatrix mettalloproteinases (MMPs) in the tissue destruction and immuneresponses related to (a) periodontal inflammation and bone loss, (b)systemic changes in MMP activity or levels in circulation, (c) skinatrophy reflecting MMP-mediated connective tissue degradation, and (III)the pharmacokinetics of relevant curcumin analogues having anelectron-withdrawing group at the C-4 carbon in this rat model ofinflammation and tissue destruction.

The purpose of the following was to establish an in vivo model ofexcessive collagen degradation to be used to test efficacy of thecompounds of the subject invention. Male Sprague-Dawley rats (typically300-400 gms of body weight), specific viral antibody free, were orderedfrom Charles River. Following general anesthesia via isofluraneanesthetic inhalation, diabetes was induced by an I.V. administration ofstreptozotocin (STZ) (50* mg/kg body weight) (Sigma Chemical Co., StLouis, Mo., USA) diluted in 0.9% citrated saline (pH 4.5), after 12hours of fasting. After the streptozotocin injection, the animals weregiven free access to water and food. Diabetic status were confirmedweekly using a glucose test strip (Tes-Tape, Eli Lilly), whichrevealed >2% glucose in the urine of the STZ-injected animals within thefirst 24-48 hours after injection of streptozotocin. In this test, thetest-paper color changes to dark brown which indicates glycosuria.During the experimental period, as expected, all the diabetic ratsdemonstrated polydipsia (excessive drinking), polyphagia (excessiveeating), polyuria (excessive urination), loss of body weight, andhyperglycemia.

A 25% mortality rate is observed after a single injection of a dose ofup to 70 mg/kg body weight of streptozotocin. This mortality rate hasbeen taken into account when determining the number of rats required perexperimental groups. Normal control animals are inoculated with anequivalent volume of citrate buffer using the same route as the diabetesinduced animals.

On day 21, animals were euthanized via CO₂ inhalation. Blood sampleswere collected by cardiac puncture into vacutainer tubes containingliquaemin sodium citrate (Liquemine, La Roche Ltd, Basel Switerland).After centrifugation, plasma was separated and aliquoted and stored at−80° C. for future analysis. A plasma glucose level of greater than 300mg/dl confirmed the presence of diabetes.

Gingival samples were collected and pooled from each group forextraction, followed by partial purification and analysis of collagenaseand gelatinase in the extracts. Spleen, heart, and salivary glands wereremoved for either histological evaluation, phenotypes of lymphocyticcells, analysis of cytokine/chemokine expression. Dorsal skin biopsyabout 2×2 inches were collected and immediately frozen at in −80° C. forcollagen content and solubility analysis. All the tissues were frozen in−80° C., and the biochemical analysis are pursued in due course. Bothupper and lower jaws were removed defleshed and stained for morphometricanalysis of bone loss under a dissecting microscope (20×). In futureexperiments, loss of bone volume may be assessed by micro CT scan.

5 controls and 8 diabetic rats (after a single i.v. injection ofstreptozotocin at 50 mg/kg body weight) were sacrificed on day 21. Bodyweight and blood glucose levels were analyzed and results are shown inFIGS. 3 and 4.

The effect of matrix metalloproteinases (MMPs) in the tissue destructionand immune responses related to periodontal inflammation, the effect ofprophylactic and/or therapeutic treatment on matrix metalloproteinases(MMPs) in the tissue destruction and immune responses related toperiodontal inflammation and bone loss, as well as other tissueresponses, and the pharmacokinetics of are examined by administration ofrelevant curcumin analogues having an electron-withdrawing group at theC-4 carbon in this rat model of inflammation and correlating theobserved changes.

Example 7

In Vitro Inhibition of MMP-8

Using the same experimental conditions described in Example 2, resultsindicated that compound 3 (Table 5), another curcumin derivative, ismore potent as a collagenase or MMP inhibitor than compound 1. Asdescribed hereinabove, compound 1 (a methoxycarbonyl curcumin) was equalin potency to 1,10-phenthroline (a zinc chelator typically used to blockcollagenase activity assays in vitro; phenanthroline is a toxic compoundnot suitable for use in vivo) as an inhibitor of human collagenase, andmore potent than natural curcumin in vitro. Moreover, compound 3 is moresoluble in aqueous solutions than compound 1 which, in turn, is moresoluble than the insoluble natural product curcumin.

TABLE 5 Structure of Compounds 3 and 4. Compound Structure

Compound 3

Compound 4

Using the same technique as in Example 2, the synthetic octapeptidesubstrate, containing the collagenase-susceptible glycine-isoleucinepeptide bond, was incubated (37° C.) with commercially-availablechromatrographically-purified human neutrophil collagenase (MMP-8) inthe presence of 1 mM Ca⁺⁺ and the tripeptide degradation fragment andundegraded substrate were separated and measured by HPLC. Compound 3 wasadded at different final concentrations ranging from 5-500 μM and the %inhibition of the collagenase activity was calculated. In thisexperiment, compound 3 was found to inhibit 50% of the collagenaseactivity (IC₅₀ ≦5 μM) at about half the concentration required forcompound 1 (IC₅₀=10 μM) (FIG. 5).

Example 8

In Vitro Inhibition of MMP-9

The ability of 1,10-phenanthroline, curcumin, compound 1, compound 3,and compound 4 (see Table 5) to inhibit a different MMP, 92 kDagelatinase or MMP-9, under the same experimental conditions describedhereinabove for human leukocyte collagenase (MMP-8) was investigated.Once again compound 3 showed the lowest IC₅₀ (6 μM) as an MMP inhibitor,this time against MMP-9 (human leukocyte gelatinase), and again had alower IC₅₀ than 1,10-phenanthroline (9 μM). All threechemically-modified curcumins, compound 1, compound 4 and compound 3,again showed lower IC₅₀ values (6-17 μM) than the natural curcumin(IC₅₀=29 μM) and the latter compound, even at a very high concentration(100 μM) was only able to inhibit degradation of the gelatinasesubstrate by 58%. In contrast all three chemically-modified curcumins at100 μm final concentration inhibited gelatinase activity by 68-100%(FIG. 6 and Table 6).

TABLE 6 Potency of Chemically-Modified Curcumins as MMP-9 InhibitorsMaximum Inhibition Test compounds IC₅₀ (μM) At 100 μM compound1,10-phenanthroline 9 100%  Curcumin 29 58% Compound 1 16 72% Compound 417 100%  Compound 3 6 68%

Example 9

Evaluation of Compound 1 In Vivo

Compound 1 (methoxycarbonyl curcumin) was further investigated in vivoin the insulin-deficient diabetic rat model, described in Example 6. Inone study, 16 male Sprague-Dawley rats (about 375 g body wt.) weredistributed into three experimental groups: non-diabetic controls (NDCgroup; n=4 rats); rats rendered diabetic by streptozotocin injection (70mg/kg) then, 2 week later, administered vehicle (carboxymethylcellulose) alone once/day by oral gavage for 7 days (UD group; n=6); anddiabetics administered orally once/day for 7 days Compound 1 (100 mg/kg)suspended in the vehicle (D+Compound 1; n=6). As expected, the diabeticrats were severely hyperglycemic compared to the NDC group (>500 mg/dlserum glucose vs. <200 mg/dl) and the oral administration of compound 1to the diabetics had no effect on the severity of hyperglycemia (FIG.7A). However, when the plasma gelatinase levels were examined by gelatinzymography, and the lytic zones measured by densitometric analysis, bothMMP-2 (72 kDa gelatinase or gelatinaseA) and MMP-9 (92 kDa gelatinase orgelatinase B) were elevated in the UD rats compared to the NDCs, andoral administration of compound 1 reduced the excessive levels of MMPsto essentially normal levels (FIG. 7B) in spite of continuing severehyperglycemia (FIG. 7A). A similar pattern was observed forcollagenase-2 (MMP-8) in extracts of skin samples from the three groups,NDC, UD and D+Compound 1 (FIG. 8). The latter assays were carried outusing Western blot analysis with monoclonal antibodies to MMP-8.

Also of interest in this short-term treatment experiment (2 weeksdiabetes with no treatment, followed by 1-week of oral treatment withcompound 1), the diabetic adverse events (AE) (Table 7) seemed toparallel the changes, described above, in plasma and skin. The UD ratsshowed the greatest incidence & severity of AEs and treatment withcompound 1 appeared to reduce them (Table 7).

TABLE 7 Diabetic Adverse Events: Effect of Compound 1 Experimental GroupIncidence of (no. of rats per group) Adverse Events (AEs) Description ofAEs NDC (n = 4) 0/4 None UD (n = 6) 3/6 Bleeding from nose and undernails; inflamed sclera; excessive tears D plus compound 1 1/6 Minorinflamed sclera (n = 6)

Example 10

Evaluation of Compound 1 In Vivo

In a second in vivo study testing orally administered compound 1, fourgroups of rats (6 rats/group) were established including non-diabeticcontrols (NDC group), diabetics orally administered vehicle(carboxymethyl cellulose) alone once/day for 3-weeks, and diabeticsorally administered a lower (100 mg/g) or higher (500 mg/kg) oral doseof compound 1 daily over the 3-week time period. At the end of thetreatment protocol, the rats were sacrificed, blood samples werecollected, and whole skin (except over limbs) and gingiva were dissected(the skin, because of ample quantities of tissue were analyzed for eachrat separately; the gingiva, because of the tiny amounts that could beharvested per rat, were pooled for each experimental group, asprescribed previously for tetracycline studies (30). In addition, thejaw bones were collected, defleshed, and bone loss around the teethanalyzed morphometrically as described previously (42, 43).

Similar to the Example 9 in vivo experiment described above, inducingdiabetes with streptozotocin dramatically increased blood glucose (aswell as hemoglobin Alc levels) and oral administration of compound 1, atboth the lower (100 mg/kg) and higher (500 mg/kg) doses, had nosignificant effect on these diagnostic markers of the severity ofdiabetes/hyperglycemia (FIG. 9).

When the pools of gingiva from the different groups of rats wereextracted, partially purified by ammonium sulfate precipitation, and thegelatinase (MMP-2 and MMP-9) levels examined by gelatin zymography (FIG.10), NDC gingiva exhibited only 72 kDa pro-forms and lower molecularweight activated forms of MMP-2 (gelatinase A) which are generallyproduced in gingiva (and skin; FIG. 12) by fibroblasts and epithelialcells. However, inducing diabetes and hyperglycemia resulted in theappearance of 92 kDa gelatinase in the gingival tissues (ie., MMP-9 orgelatinase B) which is most often associated with inflammatory cells.This appearance of MMP-9 only in the gingiva (FIG. 10), but not the skin(FIG. 12), of the diabetic rats is likely the result of the oralbacteria inducing inflammation in the gingiva of these immune-suppressedUD rats, whereas the skin is not exposed to this onslaught of bacteriaparticularly the anaerobic gram-negative bacteria in the mouth thatcause periodontal and gingival inflammation. Of importance, when thediabetic rats were treated by the oral administration of compound 1,both the lower (100 mg/kg) and the higher (500 mg/kg) doses reducedMMP-9 to the undetectable levels seen in the gingiva of the control(NDC) rats. However, neither diabetes nor compound 1 appeared to have adetectable effect on pro- and activated-forms of MMP-2 in the gingiva(FIG. 10).

When a key inflammatory mediator in the gingiva, the cytokine IL-1β, wasmeasured by ELISA in the partially-purified extracts of this oral tissue(the gingiva) a similar pattern was seen (FIG. 11). Diabetes increasedthe levels of IL-1β in the gingiva by 430% compared to the level seen inthe NDC gingiva, and the higher oral dose of compound 1 reduced thiscytokine by 95% compared to the high level of IL-1β seen in the UD ratgingiva. The lower dose of compound 1 appeared to reduce IL-1β by about18%. Concerning the effect of this compound on cell signaling pathways,which can modulate the expression of pro-inflammatory cytokines such asIL-1β, compounds of the present invention, including compound 1, caninhibit NFκB phosphorylation/activation in human monocytes.

Example 11

Effect of Compound 1 on Alveolar Bone Loss

Alveolar bone loss (the signature pathologic event in inflammatoryperiodontal disease) was assessed in the defleshed jaws of the compound1-treated (TX) and untreated rats. The results are summarized in FIG.13.

Briefly, little or no difference in bone loss was seen comparing theuntreated non-diabetic and untreated diabetic rat jaws (enhanced boneloss has been observed in diabetic rats previously when the duration ofthe hyperglycemia was greater than the 3-week experiment describedherein). In this regard, both untreated groups of rats showed alveolarbone loss scores of approximately 1.5 (see FIG. 13). In contrast, whenthe diabetics were orally administered compound 1, the lower dose (100mg/kg) produced about a 60%, statistically significant (p<0.05)reduction in bone loss compared to the untreated groups whereas the veryhigh oral dose (500 mg/kg) was less effective.

These data indicate that the oral administration of compound 1 may beeffective in reducing inflammatory- and tissue destructive-mediators ofperiodontal disease in this rat model of diabetes. The skin extracts didnot exhibit MMP-9, only MMP-2 (gelatin zymography), and the elevatedlevel of MMP-2 (assessed by ELISA) in the skin of the UD rats wasreduced by 28% after treatment with both low and high doses of the testdrug.

The lower dose of compound 1 is safer and more effective than the higherdose, and the carboxymethylcellulose vehicle, rather than theN-methylglucamine (the latter is more effective in solubilizingcompound 1) is better for the diabetic rats because the glucamine mayadversely effect the glucose metabolism of these animals.

Example 12

Evaluation of Compound 1 on hydrogen peroxide induced lactatedehydrogenase release and doxorubicin mediated caspase-3 activity inneonatal rat cardiac myocytes

Test System: Neonatal Rat Ventricular Myocytes

Test compounds and concentrations (MMP inhibitors, MMPi):

-   -   1. GM-6001: 10 μM    -   2. ONO-4817: 10 μM    -   3. Compound 1: 10 μM

The compounds were freshly prepared in DMSO (Sigma) and the final DMSOconcentration did not exceed 0.1%. In pilot studies 0.1% DMSO did nothave any effect on any of the parameters investigated.

Assays:

1. Lactate Dehydrogenase (LDH) Release

Neonatal rat ventricular myocytes, serum starved for 24 h, were treatedwith the inhibitors 1 h prior to the addition of 100 μM of hydrogenperoxide (Sigma). LDH release in the conditioned media was quantifiedusing CytoTox-ONE Homogeneous Membrane Integrity Assay reagent (Promega)at the indicated time points.

2. Caspase-3 Activity

Neonatal rat ventricular myocytes, serum starved for 24 h, were treatedwith the inhibitors 1 h prior to the addition of 500 nM of doxorubicin(Sigma). At the indicated time points cells were washed with ice-coldphosphate buffered saline and lysed using RIPA buffer (Pierce)containing protease and phosphatase inhibitors (Sigma). Caspase-3activity in the cell lysates was measured by incubation with7-amino-4-trifluoromethyl coumarin (Enzo).

Hydrogen peroxide (100 μM) induced LDH release was significantlyattenuated by compound 1 at 6 h (32904±3237 hydrogen peroxide vs.24286±930 compound 1, p<0.05, One-way-ANOVA followed by Dunnett'sposthoc) (FIG. 14). A less dramatic effect of compound 1 on LDH releasewas observed at 24 h (FIG. 15). Other MMP inhibitors (GM-6001 orONO-4817) did not affect LDH release at the tested concentrations in thecurrent experimental conditions.

None of the MMP inhibitors, including compound 1, negatively modulatecaspase-3 activity mediated by doxorubicin (500 nM) at any of the timepoints at the tested concentrations in the current experimentalconditions (FIGS. 16 and 17).

Example 13

Effect of Compound 1 on Inflammatory Disease and Tissue Degradation

Full thickness cores of bovine articular cartilage [4 mm dia.] wereharvested and equilibrated in tissue culture for 48 hours prior to anymanipulations. All cartilage plugs were then incubated with S-35 labeledsulfate in the media for 24 hours to label all aggrecan molecules in asteady state manner. The cartilage plugs were then allocated to one ofthe following groups: control, with normal media, control with mediacontaining interleukin 1 beta [IL-1β, 10 ng/ml]. IL-1 is a cytokine thatis common to inflammatory cells and present in inflammatoryenvironments. Exposure of cartilage to IL-1 results in a degradativeprocess leading to loss of aggrecan molecules, which are an importantmatrix component responsible for maintaining the mechanical propertiesof the tissue. The loss of aggrecan is reflected in the amount of S-35label present in the media after challenge with IL-1. IL-1 exposure isan indirect method for mimicking osteoarthritis. The other groupsconsisted of cartilage plugs which contained one of several novelderivatives of curcumin [10 um]—compound 1 and compound 5 (Table 5).These were also incubated with IL-1β.

The results at 24 hours showed a significant decrease in loss ofaggrecan, reflected by S-35 present in the media by all cartilage plugstreated with the experimental compounds. Control IL-1 treated cartilagedemonstrated significant loss of aggrecan compared to the experimentalcompounds and media alone control (FIG. 18). The same trends continuedfor 48 and 72 hours (FIGS. 19 and 20). The same trends were alsoobserved for 24-hour pretreatment+24-hour treatment (FIGS. 21 and 22).

Example 14

MMP Inhibitory Activity of Amide-Containing Curcumin Derivatives

Chemically-modified curcumins having a carbonyl-amide-phenol group atcarbon 4 have been synthesized and exhibit greater solubility thancurcumin. These amide-containing chemically-modified curcumins are weretested for their ability to inhibit chromatographically-purifiedMMPs—MMP-2 and MMP-13—using the same assay described previously inExample 2, ie., the degradation of the MMP-susceptible, syntheticoctapeptide and the measurement of the tripeptide degradation fragmentand residual substrate by HPLC (37° C., 1 mM Ca⁺⁺). As shown in Table 9,curcumin was less potent as an inhibitor of MMP-2 than the Zn-chelatingagent, 1,10-phenanthroline (higher IC₅₀, and less inhibitory activitiesat 100 μM concentration). Compound 1 (based on in vitro, cell culture,and in vivo efficacy) was again most potent as an MMP inhibitor (thistime against MMP-2, showing the lowest IC₅₀ ratio: test compound vs.standard (ratio=0.7) and inhibiting MMP-2 activity by 78% at 100 μMconcentration.

TABLE 8 Structure of amide-containing curcumin compounds Compoundstructure

Compound 6

Compound 7

Compound 8

In contrast, compounds 4 and 3, which are not amide compounds but whichshowed excellent potency as inhibitors of MMP-9 in Example 8, showedvery different levels of efficacy against MMP-2 (Table 9).

TABLE 9 MMP Inhibitory Potency of Amide- Containing Curcumin DerivativesMMP-2 MMP-13 Compound Maximum Inhi- Maximum Inhi- Tested Ratio* bition @100 μM Ratio* bition @ 100 μM 1,10- 1.0 60% 1.0 100%  phenanthroline(standard) Curcumin 1.2 54% 27.5 53% Compound 1 0.7 78% 3.8 69% Compound4 >3.6 36% 62.5 50% Compound 3 1.0 60% 2.0 76% Compound 6⁺⁺ 1.2 53% <0.3100%  Compound 7⁺⁺ 1.8 45% <0.3 77% Compound 8⁺⁺ — — 11.3 53%⁺⁺amide-containing curcumin derivatives *Ratio = IC₅₀ test compound/IC₅₀standard

Compound 4 was less effective than the standard, 1,10-phenanthroline,while compound 3 was equal in efficacy to 1,10-phenanthroline. Of thethree amide-containing compounds tested, compounds 6, 7, and 8, theefficacy of MMP-2 inhibition was as follows: compound 6>compound7>compound 8. Although compound 6 showed similar efficacy as curcuminwhen comparing MMP-2 inhibitory potency, the amide-containing compoundsare much more soluble than the famously insoluble curcumin.

The amide-containing curcumin derivatives are much more potentinhibitors of MMP-13 (Collagenase-3) than curcumin and even more potentthan compound 1 (Table 9 and 10).

It should be noted that the tetracyclines, which resulted in twoFDA-approved drugs for chronic inflammatory diseases, were veryeffective as inhibitors of MMP-9 and MMP-13 and much less effectiveagainst MMP-2 (see Brown et al, 2004; Sorsa et al). Compounds 1, 6, and7 fit such a profile (Table 10) and, therefore, are expected to beeffective for use in treating chronic inflammatory diseases.

TABLE 10 Concentration of Curcumin Derivatives Required to Inhibit 50%of Enzyme Activity (IC₅₀ values) IC₅₀ values (μM; Inhibition ofDifferent MMPs) Compound Tested MMP-2 (μM) MMP-13 (μM)1,10-phenanthroline 70 4 Curcumin 85 110 Compound 1 48 15 Compound4 >250*  250 Compound 3 70 8 Compound 6⁺⁺ 85 <1 Compound 7⁺⁺ 125  <1Compound 8⁺⁺ Unmeasurable* 45 ⁺⁺more soluble amide-containing compounds*evidence of precipitation

Example 15

Evaluation of Compound 1 in a Cell Culture Model of Cancer

Compound 1 was evaluated in a cell culture model of cancer. In brief,PC-3 human prostate cancer cells were incubated in serum-free media for48 hr (37° C., pH7.6) in the presence or absence of EGF and compound 1was added to the cultures at a final concentration of 0, 10, and 20 μM.After the incubation, aliquots of the conditioned media were assayed forMMP-9 (gelatinase B) by either of two techniques, gelatin zymography (inthe presence of 2 or 10 mMCa⁺⁺) and Western blot (both assays usedpurified MMP-9, 92 kDa gelatinase, as a standard). As shown in FIGS.23-25, the 10 μM and 20 μM concentrations of compound 1 inhibited theproduction and/or activity of 9 generated by the human prostate cancercells.

Discussion

While investigations have been conducted to improve curcumin through thesynthesis of curcumin-based analogues and derivatives (14-18), previousefforts did not appreciate that installation of an electron-withdrawinggroup on the C-4 carbon (see FIG. 1) of curcumin and its analoguesimproves a number of pharmacologically important characteristics of suchcompounds.

As alluded to earlier, the biological activity of curcumin and itsanalogues is attributed to their ability to access and bind zinc ions.The discovery of the enhancement of zinc binding affinity through theinstallation of electron-donating and electron-withdrawing groups atstrategic locations, namely the C-4 carbon and the aryl rings, on thecurcumin skeleton is important for enhancing biological activity. Inparticular, the incorporation of an electron-withdrawing group on theC-4 carbon is shown to be essential for the improvement of zinc bindingaffinity, which leads to an enhancement in biological activity.

Published work which examined curcumin analog compounds having anelectron-withdrawing group indicated contrasting effects of such agroup. Lin and co-workers reported lower cytoxicity (higher IC₅₀), ie. areduction of activity, with a curcumin analogue having an acetyl groupat the C-4 position (compound 49 in ref. 10) in comparison with that ofcurcumin (compound 1 in ref. 14) when tested against human prostatecancer cells (14). On the contrary, Shih et al. reported reducedandrogen receptor (AR) expression within cancer cells, ie. highercytotoxicity, with a dimethylcurcumin analogue having a substitutedphenylpropenal moiety (compound ASC-JM4 in ref. 11b) at lowconcentrations when compared to dimethylcurcumin (compound ASC-J9 inref. 15b). However, Shih et al. did not publish on further curcuminshaving other substituents at the C-4 carbon. These studies indicate thatresearchers have not appreciated the importance of anelectron-withdrawing group at the C-4 carbon of curcumin and itsanalogues.

As described herein, the placement of an electron-withdrawing group atthe C-4 carbon of curcumin and curcumin analogues confers severaladvantages, such as improved water solubility, improved metal bindingability, and improved biological activity when compared to curcumin.Without wishing to be bound by theory, it is believed that the presenceof an electron-withdrawing group at the C-4 position of curcumin andcurcumin analogues stabilizes the enol form of the compound as well asthe enolate formed from deprotonation at the C-4 carbon, therebyfacilitating water solubility and chelation of metal cations, such asZn²⁺, by the resulting curcumin enolate. Accordingly, other curcuminanalogues having electron-withdrawing groups at the C-4 carbon possesssuch prope and function in a similar manner.

Compounds of the subject invention are tested and exhibit activityconsistent with those of the foregoing examples.

Specifically, the compounds of the subject invention are useful for theinhibition of matrix metalloproteinase activity as well as theinhibition of both cytokine production and NFκB activation in vivo andin vitro, and are useful for treating pathologies in subjects arisingfrom matrix metalloproteinase activity, growth factor activity, cytokineproduction, and/or NFκB activation.

Although numerous experimental agents have been developed over the yearswith excellent potency as inhibitors of collagenases (MMP-1, MMP-8 andMMP-13) and other MMPs (e.g., the gelatinases, MMP-2 and MMP-9; thestromelysins, MMP-3 and MMP-10) (8), the only MMP-inhibitor (MMPI) drugapproved by the U.S. FDA and regulatory agencies in Europe and Canada isa NON-ANTIBIOTIC formulation of doxycycline (subantimicrobial-dosedoxycycline, SDD) called Periostat® marketed for the treatment of themost common of all chronic inflammatory diseases, periodontitis, whichinvolves the destruction of collagen and other connective tissues in thegingiva, periodontal ligament, and bone, as mentioned previously (4).Oracea®, a sustained-release SDD, also administered systemically by theoral route, was recently approved by the US FDA and in Europe for thetreatment of a chronic inflammatory skin condition, rosacea.

Several factors explain, at least in part, this difficulty in bringingMMPI drugs to clinical application: (1) It is recognized that MMPs playa role in various physiologic (not just pathologic) processes, such asprocessing anti-inflammatory cytokines and chemokines as well asmodulating growth factors or cell surface receptors, and regulatingcellular proliferation and apoptotic and immune responses (2, 3, 27).Therefore, the goal of MMPI therapy must be to dampen or modulatepathologic levels of MMPs, NOT to excessively inhibit them (2, 4, 27);and (2) Based on past experience with SDD, and other drug-developmentstrategies, it may be desirable to chemically-modify compounds whichincorporate the same or similar active sites for MMP-inhibition, whichare derived from agents with a long history of safety in humans since,for chronic diseases, they are likely to have to be administered forlong periods of time. Considering the MMPI drugs, Periostat® andOracea®, these were based on the well-known drug, doxycycline, atetracycline which was deliberately titrated down to subantimicrobialconcentrations in the circulation, after oral administration, so thatthey would not produce antibiotic side-effects during long-termadministration, but which would retain the Ca⁺⁺ and Zn⁺⁺ site atcarbon-11 and carbon-12 to inhibit collagenases and gelatinases, as wellas other pleiotropic benefits of those drugs (see ref. 4 for review).However, because of the concern for the side-effects of alltetracyclines including doxycycline, only a narrow (perhaps sub-optimal)range of non-antimicrobial blood levels of the drug (e.g., 0.3-0.8 μgml) could be considered therapeutically useful because of the concernfor the emergence of antibiotic-resistant bacteria at higher bloodlevels (and other concerns, such as increased sensitivity to sunburn).

The capability to down-regulate MMP production and activity, but not toinhibit these enzymes completely, has a significant impact on severaldiseases which are chronic afflictions particularly in adults and olderage groups. As described herein, the compounds and compositions of thepresent invention inhibit the production of a variety ofpro-inflammatory cytokines (e.g., IL-1β, TNFα, IL-6) which are also keyparticipants in the pathogenesis of these chronic diseases. Thesediseases include, but are not limited to, impaired wound healing andother skin conditions (e.g., psoriasis (44)), periodontitis, arthritis,cardiovascular disease, osteoporosis, acute respiratory distresssyndrome, and cancer. The development of an effective non-toxicmodulator of MMPs and cytokines constitutes a significant advancement inpharmacotherapeutics. The safety and efficacy of the compounds of thepresent invention in vivo in diabetic complications in skin (e.g., skinaging/atrophy), and in inflammatory gingival disease and periodontalbone loss, and in cell and tissue culture models of arthritis andcardiac myocyte dysfunction and MMPs produced by prostate cancer cellsare shown herein.

Using a severely hyperglycemic type I diabetic rat model, which producesincreased levels of MMPs and pro-inflammatory cytokines compared tonon-diabetic controls, the oral administration of compound 1 has beenfound to: (i) reduce MMP-2 and MMP-9 in plasma, (ii) reduce MMP-2 andMMP-8 in skin, and (iii) reduce MMP-9 and IL-1β in gingiva. Moreover,oral administration of compound 1 also reduced pathologic alveolar boneloss which, together with elevated MMPs and cytokines, is a signatureevent in both diabetes and periodontitis. All of these beneficialeffects were accompanied by an apparent improvement in adverse eventsproduced by the severe diabetic condition. Diabetic complications, notthe severity of diabetes per se, were improved.

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What is claimed is:
 1. A compound having the structure

wherein bond α and β are each, independently, present or absent; X isCR₅ or N; Y is CR₁₀ or N; R₁ is —SR₁₂, —SO₂R₁₃, —COR₁₄, —CSR₁₄,—C(═NR₁₂)R₁₄, —C(═NH)R₁₄, —SOR₁₂, —POR₁₂, —P(═O)(OR₁₂) (OR₁₃), or—P(OR₁₂).(OR₁₃), wherein R₁₂ is C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, aryl, heteroaryl, or heterocyclyl; R₁₃ is H, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl; R₁₄ is C₂₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroaryl, heterocyclyl, methoxy,—OR₁₅, or —NR₁₆R₁₇, wherein R₁₅ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl; R₁₆ and R₁₇ are each, independently, H, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, or heterocyclyl; R₂, R₃, R₄,R₅, R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are each independently, H, halogen,—NO₂, —CN, —NR₂₈R₂₉, —NHR₂₈R₂₉ ⁺, —SR₂₈, —SO₂R₂₈, —OR₂₈, —CO₂R₂₈, CF₃,C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, aryl, heteroaryl, orheterocyclyl; wherein R₂₈ and R₂₉ are each, H, CF₃, C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, or —C(═O)-heterocyclyl; and wherein eachoccurrence of alkyl, alkenyl, or alkynyl is branched or unbranched,unsubstituted or substituted; or a salt thereof.
 2. The compound ofclaim 1, wherein R₁ is —SR₁₂, —SO₂R₁₃, —COR₁₄, —CSR₁₄, —C(═NR₁₂)R₁₄,—C(═NH)R₁₄, or —SOR₁₂.
 3. A compound of claim 2, wherein R₁ is —COR₁₄,—CSR₁₄, —C(═NR₁₂)R₁₄, or —C(═NH)R₁₄.
 4. The compound of claim 1, havingthe structure

or a salt thereof.
 5. The compound of claim 1 having the structure:

or a salt thereof.
 6. The compound of claim 1 having the structure:

wherein R₃, R₄, R₈, and R₉ are each, independently, H, —OCH₃, or —OH;and R₁₄ is methoxy or —N(CH₃)₂, or a salt thereof.
 7. The compound ofclaim 1 having the structure:

or salt thereof.
 8. The compound of claim 1 having the structure:

or a salt thereof.
 9. The compound of claim 1 having the structure:

or a salt thereof.
 10. The compound of claim 1 having the structure:

or a salt thereof.
 11. The compound of claim 1 having the structure:

or a salt thereof.
 12. The compound of claim 1 having the structure:

or a salt thereof.
 13. A compound having the structure:

or a salt thereof.
 14. A compound having the structure:

or a salt thereof.